Detection of Enterotoxin Genes in Bacillus species Isolated from Cassava
Processing Environment in Nigeria

Nkem      Torimiro; Oluwafemi   B.   Daramola; Richard   K.   Omole; Ifeyimika   Z.   Adesina

doi:10.2174/2666121702666220112101938

Abstract

Introduction: The health challenges associated with cassava products as a common staple food for approximately 70 % of Africans and part of Asia pose a looming danger due to Bacillus enterotoxins’ presence in the processing environment.

Objective: This study investigated the presence of enterotoxigenic genes, namely, Bacillus cereus enterotoxin T (bceT), hemolysin bl (hblC, hblD), and non-haemolytic enterotoxin (nheA, nheB, and nheC) from Bacillus species isolated from soil of cassava processing environment.

Methods: Soil samples from 20 cassava processing sites in Ile-Ife and Modakeke, Nigeria, were collected and cultured on nutrient agar at 37ºC for 24 hours. Colonies phenotypically identified as Bacillus were identified using Bacillus-specific 16S rRNA-targeted PCR technique. Screened Bacillus spp were assessed for the presence of enterotoxigenic genes using PCR with previously reported primers.

Results: A total of 100 Bacillus isolates were selected from this study, with Bacillus macerans (33 %) showing the highest frequency of occurrence among the identified species, however, 74 isolates were molecularly confirmed as Bacillus. Amongst the 74 molecularly confirmed Bacillus isolates, 28 (37.84 %), 35 (47.30 %), and 37 (50 %) had nhe, hbl, and bceT genes, respectively. Investigation showed that 42 (56.76 %) of the Bacillus species encoded at least one of the screened enterotoxin genes.

Conclusion: The presence of these 3 sets of enterotoxin genes in Bacillus isolated from cassava processing sites calls for immediate attention as they could be pivotal in the release of toxins in cassava products, causing lethal effects via consumption. This study demonstrates the possibility of foodborne disease outbreaks in Bacillus toxin-laden cassava products processed under unhygienic conditions.

Keywords: Bacillus toxins, food safety, foodborne disease, enterotoxigenic genes, cassava, hygienic practices.

Graphical Abstract

[1] 
Reincke K, Vilvert E, Fasse A, Graef F, Sieber S, Lana MA. Key factors influencing food security of smallholder farmers in Tanzania and the role of cassava as a strategic crop. Food Secur  2018; 10(4): 911-24.
[http://dx.doi.org/10.1007/s12571-018-0814-3] 
[2] 
Ogunyinka O, Oguntuase A. Analysis of cassava production and processing by various groups in support of cassava value chain in the south west of Nigeria. J Food Agric Sci  2020; 9(1): 11-9.
[http://dx.doi.org/10.5897/ISABB-JFAS2020.0113] 
[3] 
Oyewole BO. Cassava processing in AfricaApplications of biotechnology to fermented foods: report of an ad hoc panel of the board on science and technology for international development. United States: National Academies Press 1992.
[http://dx.doi.org/10.17226/1939] 
[4] 
Kouamé AK, Bouatenin MJK, Djéni TN, Dje KM. Identification of hazards and critical control points during attiéké (a fermented cassava product) process in Côte d’Ivoire. Lett Appl Microbiol  2020; 70(2): 87-94.
[http://dx.doi.org/10.1111/lam.13247] [PMID: 31705681] 
[5] 
Shittu TA, Alimi BA, Wahab B, Sanni LO, Abass AB. Cassava flour and starch: Processing technology and utilization. Tropical roots and tubers, production, processing and technology.  New York: John Wiley and Sons Ltd. 2016; pp. 415-50.
[http://dx.doi.org/10.1002/9781118992739.ch10a] 
[6] 
Avşar C, Koyuncu H, Aras ES. Isolation and molecular characterization of Bacillus spp isolated from soil for production of industrial enzymes. Biological and Chemical Research.  NY, USA: Science Signpost Publishing 2017; pp. 72-86.
[7] 
Felske A, Wolterink A, Van Lis R, Akkermans AD. Phylogeny of the main bacterial 16S rRNA sequences in Drentse A grassland soils (The Netherlands). Appl Environ Microbiol  1998; 64(3): 871-9.
[http://dx.doi.org/10.1128/AEM.64.3.871-879.1998] [PMID: 9501427] 
[8] 
Liszka MJ, Clark ME, Schneider E, Clark DS. Nature versus nurture: developing enzymes that function under extreme conditions. Annu Rev Chem Biomol Eng  2012; 3: 77-102.
[http://dx.doi.org/10.1146/annurev-chembioeng-061010-114239] [PMID: 22468597] 
[9] 
Felske AD, Heyrman J, Balcaen A, de Vos P. Multiplex PCR screening of soil isolates for novel Bacillus-related lineages. J Microbiol Methods  2003; 55(2): 447-58.
[http://dx.doi.org/10.1016/S0167-7012(03)00191-X] [PMID: 14529967] 
[10] 
Aygan A, Sariturk S, Kostekci S, Tanis H. Production and characterization of alkaliphilic alpha-amylase from Bacillus subtilis A10 isolated from soils of Kahramanmaras, Turkey. Afr J Microbiol Res  2014; 21: 2168-73.
[http://dx.doi.org/10.5897/AJMR2013.6586] 
[11] 
Rasko DA, Altherr MR, Han CS, Ravel J. Genomics of the Bacillus cereus group of organisms. FEMS Microbiol Rev  2005; 29(2): 303-29.
[http://dx.doi.org/10.1016/j.fmrre.2004.12.005] [PMID: 15808746] 
[12] 
Deb P, Talukdar SA, Mohsina K, Sarker PK, Sayem SA. Production and partial characterization of extracellular amylase enzyme from Bacillus amyloliquefaciens P-001. Springerplus  2013; 2(1): 154.
[http://dx.doi.org/10.1186/2193-1801-2-154] [PMID: 23626928] 
[13] 
Fricker M, Messelhäusser U, Busch U, Scherer S, Ehling-Schulz M. Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks. Appl Environ Microbiol  2007; 73(6): 1892-8.
[http://dx.doi.org/10.1128/AEM.02219-06] [PMID: 17259359] 
[14] 
Horii T, Notake S, Tamai K, Yanagisawa H. Bacillus cereus from blood cultures: Virulence genes, antimicrobial susceptibility and risk factors for blood stream infection. FEMS Immunol Med Microbiol  2011; 63(2): 202-9.
[http://dx.doi.org/10.1111/j.1574-695X.2011.00842.x] [PMID: 22077223] 
[15] 
Kim MJ, Han JK, Park JS, et al. Various enterotoxin and other virulence factor genes widespread among Bacillus cereus and Bacillus thuringiensis strains. J Microbiol Biotechnol  2015; 25(6): 872-9.
[http://dx.doi.org/10.4014/jmb.1502.02003] [PMID: 25791850] 
[16] 
Rowan NJ, Deans K, Anderson JG, Gemmell CG, Hunter IS, Chaithong T. Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae. Appl Environ Microbiol  2001; 67(9): 3873-81.
[http://dx.doi.org/10.1128/AEM.67.9.3873-3881.2001] [PMID: 11525980] 
[17] 
Stenfors Arnesen LP, Fagerlund A, Granum PE. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev  2008; 32(4): 579-606.
[http://dx.doi.org/10.1111/j.1574-6976.2008.00112.x] [PMID: 18422617] 
[18] 
Ehling-Schulz M, Vukov N, Schulz A, et al. Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus. Appl Environ Microbiol  2005; 71(1): 105-13.
[http://dx.doi.org/10.1128/AEM.71.1.105-113.2005] [PMID: 15640177] 
[19] 
Zuberovic Muratovic A, Tröger R, Granelli K, Hellenäs KE. Quantitative analysis of cereulide toxin from Bacillus cereus in rice and pasta using synthetic cereulide standard and 13C6-cereulide standard - a short validation study. Toxins (Basel)  2014; 6(12): 3326-35.
[http://dx.doi.org/10.3390/toxins6123326] [PMID: 25514093] 
[20] 
Tran SL, Guillemet E, Ngo-Camus M, et al. Haemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages. Cell Microbiol  2011; 13(1): 92-108.
[http://dx.doi.org/10.1111/j.1462-5822.2010.01522.x] [PMID: 20731668] 
[21] 
Montanari G, Borsari A, Chiavari C, Ferri G, Zambonelli C, Grazia L. Morphological and phenotypical characterization of Bacillus sporothermodurans. J Appl Microbiol  2004; 97(4): 802-9.
[http://dx.doi.org/10.1111/j.1365-2672.2004.02371.x] [PMID: 15357730] 
[22] 
Yamagishi J, Sato Y, Shinozaki N, et al. Comparison of boiling and robotics automation method in DNA extraction for metagenomic sequencing of human oral microbes. PLoS One  2016; 11(4): e0154389.
[http://dx.doi.org/10.1371/journal.pone.0154389] [PMID: 27104353] 
[23] 
Ouoba LII, Parkouda C, Diawara B, Scotti C, Varnam AH. Identification of bacillus spp. from bikalga, fermented seeds of Hibiscus sabdariffa: phenotypic and genotypic characterization. J Appl Microbiol  2008; 104(1): 122-31.
[http://dx.doi.org/10.1111/j.1365-2672.2007.03550.x] [PMID: 17922831] 
[24] 
Ahaotu I, Anyogu A, Njoku OH, Odu NN, Sutherland JP, Ouoba LII. Molecular identification and safety of Bacillus species involved in the fermentation of African oil beans (Pentaclethra macrophylla Benth) for production of Ugba. Int J Food Microbiol  2013; 162(1): 95-104.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2013.01.001] [PMID: 23376783] 
[25] 
Elkhalil EA, Gaffar FY, Marmar A, Osman HA. Isolation and molecular characterization of cellulolytic Bacillus isolates from soil and compost. Am J Microbiol Res  2015; 2: 55-8.
[http://dx.doi.org/10.12691/ajmr-3-2-2] 
[26] 
Owusu-Kwarteng J, Wuni A, Akabanda F, Tano-Debrah K, Jespersen L. Prevalence, virulence factor genes and antibiotic resistance of Bacillus cereus sensu lato isolated from dairy farms and traditional dairy products. BMC Microbiol  2017; 17(1): 65.
[http://dx.doi.org/10.1186/s12866-017-0975-9] [PMID: 28288581] 
[27] 
Rowan NJ, Caldow G, Gemmell CG, Hunter IS. Production of diarrheal enterotoxins and other potential virulence factors by veterinary isolates of bacillus species associated with nongastrointestinal infections. Appl Environ Microbiol  2003; 69(4): 2372-6.
[http://dx.doi.org/10.1128/AEM.69.4.2372-2376.2003] [PMID: 12676723] 
[28] 
Reis AL, Montanhini MT, Bittencourt JV, Destro MT, Bersot LS. Gene detection and toxin production evaluation of hemolysin BL of Bacillus cereus isolated from milk and dairy products marketed in Brazil. Braz J Microbiol  2014; 44(4): 1195-8.
[http://dx.doi.org/10.1590/S1517-83822013000400024] [PMID: 24688511] 
[29] 
Brézillon C, Haustant M, Dupke S, et al. Capsules, toxins and AtxA as virulence factors of emerging Bacillus cereus biovar anthracis. PLoS Negl Trop Dis  2015; 9(4): e0003455.
[http://dx.doi.org/10.1371/journal.pntd.0003455] [PMID: 25830379] 

Rights & Permissions Print Cite

Article Metrics

10

1

DOI https://dx.doi.org/10.2174/2666121702666220112101938	Print ISSN 2666-1217
Publisher Name Bentham Science Publisher	Online ISSN 2666-1225

Venoms and Toxins

Detection of Enterotoxin Genes in Bacillus species Isolated from Cassava Processing Environment in Nigeria

Abstract Play Pause

Graphical Abstract

Abstract