The steadily increasing use of antibiotics with the aim of controlling microorganisms which may damage food or infective agents for human, plants or animals led to the emergence of resistant microorganisms to a wide range of antimicrobial agents . It is estimated that in Europe, approximately 25 000 persons are deceased on an annual basis because of infections caused by bacteria which are antibiotic resistant . The emergence of drug-resistant bacteria requires us to search for and develop new antimicrobial agents. One viable alternative could be the use of bacteriocins [3–5]. Bacteriocins are small peptides (approximately between 12 and 70 amino acids) with antimicrobial activity, synthesized by prokaryotes. These peptides originally emerged as adaptation mechanisms , which allow them to compete for nutrients and space in their habitat, inhibiting growth within the members of the same producer species or members of different bacterial genera [4,7]. Lactic acid bacteria have been widely studied as bacteriocin producers . For example, nisin, the most-commonly studied lantibiotic, has been approved as a food preservative in more than 50 countries . In food, the addition of bacteriocins represents an excellent strategy to control pathogenic bacteria and/or food spoilers, such as Listeria monocytogenes, Clostridium botulinum, Yersinia enterocolitica, Staphylococcus aureus, Escherichia coli, Salmonella enterica subsp. enterica and Bacillus cereus, among others . Dissemination of multidrug-resistant bacteria as S. aureus, Pseudomonas aeruginosa and Enterococcus sp. represents a matter of great concern through an important increase in the hospital costs and morbimortality . During the 1970s another bacterium, Clostridium difficile, was found to be responsible for nosocomial diarrhoea . Since then, the cases have increased in number and severity in many parts of the world. The main predisposing factor is broad-spectrum antibiotics, which eradicate beneficial bacteria, leading to C. difficile overgrowth .
The emergence of its resistance to certain conventional antibiotics leads to research with a view to discovering novel therapeutic strategies. In addition to lactic acid bacteria, different members of the Bacillus sp. genus, particularly from the cereus group, such as Bacillus thuringiensis, have been studied because of their capability of producing bacteriocins. B. thuringiensis is a Gram-positive bacterium, which produces endospores and is well known for synthesizing an enormous ‘battery’ of compounds with biological activity, specially Cry, Cyt and Vip proteins, possessing biocidal activity against certain kinds of insects. Although B. thuringiensis also synthesizes proteins with antimicrobial activity . In this regard, the knowledge relating to the mode of action of several B. thuringiensis antimicrobials is still limited. The application of B. thuringiensis antimicrobials is very important within different areas such as food biopreservation, medicine and environmental care. This review describes some of the most important aspects of bacteriocins synthesized by different strains of B. thuringiensis, such as activity spectrum, molecular mass and biological activity.
Bacteriocins synthesized by B. thuringiensis
In the last few years, there has been a resurgence in interest with respect to the discovery of new treatments including the use of microorganisms as natural products. B. thuringiensis is considered a model organism for production of metabolites, which is currently employed in the biological control of plagues and agricultural crops. In 1989, Favret and Younsten  reported the detection of a peptide synthesized by a B. thuringiensis, which was subsequently named thuricin. So far, there is a report of 18 bacteriocins synthesized by different B. thuringiensis strains. Among these 18 bacteriocins are five inhibitory substances, which resemble bacteriocins known as bacteriocin-like inhibitory substance (BLIS)  (Table 1). Bacteriocins are peptides with a different molecular weight, varying from almost 1 kDa (thuricin, 0.950 kDa) to 12.4 kDa (entomocin 9). In terms of molecular weight, we propose to divide these bacteriocins, into groups A and B. Group A is composed of the bacteriocins weighing less than 5 kDa and group B which weigh more than 5 kDa. Although from a functional point of view, within group A, we can find two types: single peptide and two-component peptide. Almost all of them belong to type I and only thuricin CD and thuricin 439 belong to type II (Table 1). Another important aspect is that members of the serological subsp. entomocidus produce three different peptides, thuricin S, entomocin 110 and entomocin 9 (Table 1). On the other hand, the same peptide can be produced by different sub-species such as thuricin S, which is produced by the serological subsp. entomocidus and subsp. tolworthy (Table 1).
Gram-positive bacteria inhibited by B. thuringiensis bacteriocins
Most bacteriocins synthesized by B. thuringiensis are broad-spectrum, inhibiting several bacterial species. Some B. cereus strains synthesize toxins, which induce diarrhoea, whereas others may be responsible for causing nausea and vomiting . Another important pathogen is L. monocytogenes, which may be inhibited by different bacteriocins, amongst which include: thuricin, thuricin S, thuricni Bn1, thurincin H, entomocin 110, bacthuricin F103, thuricin 7, entomocin 9 and thuricn CD. Listeriosis is a zoonotic disease, which is rare in humans but is still extremely serious, presenting low morbility and high mortality. Generally, contamination takes place while consuming food containing L. monocytogenes, which are responsible of causing the disease . Another bacterium, methicillin-resistant Staphylococcus aureus, has become immensely important in intrahospital as a causative agent of wound infections [19,20]. Bacteriocins such as thuricin, thurincin H, bacthuricin F4 and BLIS reported by Barboza-Corona et al. , morricin 269, kurstacin 287, kenyacin 404, entomocin 420 and tolworthcin 524 may represent the best option to control this problematic bacterium (Table 2).
Gram-negative bacteria inhibited by B. thuringiensis bacteriocins
Bacteriocins including bacthuricin F4, thuricin 7, morricin 269, kurstacin 287, kenyacin 404, entomocin 420 and tolworthcin 524 have the potential to control Klebsiella pneumoniae; a Gram-negative bacillus, which lives in the human intestine as part of its microbiota [16,21,22], considered one of the most important agents in relation to nosocomial infections, producing septicaemia and urinary and respiratory tract infections . Another important microorganism is P. aeruginosa, which may be found in hospitals. This opportunistic pathogen is frequently present in extra intestinal infections, mainly in inmmunocompromised and malnourished patients . This microorganism shows resistance to almost all conventional antimicrobials , even though it is susceptible to entomocin 110 and entomocin 9 (Table 3).
Some members of the BLIS group showed inhibitory activity against Vibrio cholerae. Since ancient times, cholera has been the cause of deadly epidemics; however, nowadays its morbimortality has diminished. Nonetheless, there are certain endemic zones presenting with a high percentage of this disease, which causes acute gastroenteritis. If the disease is not correctly treated, it may even cause death  (Table 4).
Fungi inhibited by B. thuringiensis bacteriocins
Fungi may also be targeted by some bacteriocins. For instance, Bn1 bacteriocin and entomocin nine inhibit Aspergillus growth [26,27]. Food containing starch (as in the case of potatoes and bread) may stimulate its growth. In addition, it may grow within dirty air conditioning units located in buildings and houses, representing a serious problem, including in hospitals. Frequently, Aspergillus niger is present in human ear infections and in certain instances it can even infect the lungs of people who present with a weak immune system. A. niger may be toxic if ingested through infected fruits, vegetables or grains; it can produce food poisoning (single ingestion) or cancer (chronic consumption) .
Potential applications of B. thuringiensis bacteriocins
Thuricin S: This bacteriocin is heat resistant and stable in a wide range of pH conditions and inhibits not only the pathogens that are transmitted through food causing poisoning such as L. monocytogenes and B. cereus, but also a great number of Gram-negative pathogens such as S. enterica subsp. enterica and P. aeruginosa. The use of B. thuringiensis as a biological insecticide, as well as the information about the bacteriocins, like thuricin S, empower its practical use as an antagonistic agent towards phytopatogenic bacteria such as Pseudomonas syringae[29,30]. Thuricin Bn1: This peptide is synthesized by B. thuringiensis subsp. kurstaki Bn1, which presents bifunctional properties in agriculture. This strain is active against insects through a crystalline protein  and active against plant pathogenic bacteria through a bacteriocin. On the other hand, the Bacillus genus includes a great variety of species that may be safely used within industry. Example, B. thuringiensis Bn1 produces Bacteriocin Bn1, which as proteinaceous nature, can be used as a natural food preservative . Besides, bacteriocins have gained importance as natural biopreservatives for degradation control and against pathogenic bacteria transmitted by food . Thurincin H: This bacteriocin inhibits the growth of B. cereus, L. monocytogenes, Listeria innocua and Listeria ivanovii. All of these bacteria seem to affect both animals and humans . An example includes the fatal case where L. innocua was found on a hemoculture. In this situation, the patient died because of septic shock caused by cholangitis . Bacthuricin F4: This bacteriocin is stable at a wide pH range and heat treatment, which indicates that the bactericidal function can be preserved even in extreme conditions. This presents an extremely interesting characteristic for its use in the agro-industry. The study of the bacteriocin inhibition spectrum against indicator strains revealed that this bacteriocin presents a bactericidal effect against bacterial strains highly linked to B. thuringiensis and other Gram-positive bacteria. Importantly, it is highly active against B. cereus, which is one of the most virulent bacteria that may be found in agro-industrial products . Although this bacteriocin is less active against Gram-negative bacteria . It is the only bacteriocin produced by B. thuringiensis, which displays activity against K. pneumoniae.Thuricin 17: This peptide was isolated from a bacterium, which promotes plant growth , and has been demonstrated to increase plant growth . This peptide is stable in a wide range of temperatures and is biologically active between pH 1.00 and 9.25. In addition, thuricin 17 is the only B. thuringiensis bacteriocin which shows activity against E. coli. Entomocin 110: This bacteriocin has been demonstrated to be inhibitory to several Gram-positive bacteria, including L. monocytogenes, Paenibacillus larvae and other Bacillus species . P. larvae are extremely important bacteria within the apiculture sector, as they produce the American foulbrood disease, which affects bee larvae . Tochicin: This bacteriocin exhibits activity against B. cereus and certain B. thuringiensis subspecies . Although its spectrum of activity is unclear as it has not been tested against L. monocytogenes, as is the case of bacthuricin F4 it has not been tested in L. monocytogenes or S. aureus, as it happens in the case of thuricin S. For this reason, its application and potential use in biomedical treatments has not been completely defined. Thuricin F103: This bacteriocin presents a wide spectrum of inhibition against the related bacteria and different pathogens transmitted through food such as L. monocytogenes, B. cereus and the phytopatogenic bacterium Agrobacterium tumefaciens. Subsequently, Kaumon and his colleagues  demonstrated that bacthuricin F103 may be applied to raw beef meat with the purpose of avoiding contamination by L. monocytogenes. Thuricin 7: The action and inhibitory spectrum of this bacteriocin includes B. cereus and Bacillus weihenstephanensis strains (commonly associated with the deterioration of dairy products and raw milk). Such properties make thuricin 7 the perfect candidate to be used as a food antimicrobial agent in order to control Gram-positive bacterial contaminants, without interfering with lactic acid bacteria growth . Entomocin 9: This bacteriocin displays antimicrobial activity against L. monocytogenes, P. aeruginosa and several fungi. Entomocin 9 is a new bacteriocin produced by B. thuringiensis HD9 characterized as being heat stable. Lack of toxicity against Vero cells renders strain HD9 as being suitable for a safe application in antimicrobial treatments. The importance and impact of entomocin 9 makes B. thuringiensis strain HD9 an attractive microorganism in biotechnological applications as an antimicrobial agent in the food and agricultural industry . Thuricin CD: The effectiveness of thuricin CD against the clinical C. difficile isolates, especially hypervirulent PCR R027 strains, makes it extremely interesting because of its structural characteristics and potential as a therapeutic agent. The isolation and characterization of thuricin CD may represent a structural class of bacteriocins as well as potentially providing a treatment for C. difficile-associated disease . Thuricin 439:B. thuringiensis strain B439 produces thuricin 439, a small substance like bacteriocin produced as two active peptides with different molecular mass. Thuricin 439 is active against B. cereus and B. thuringiensis strains, while at the same time showing activity against L. innocua 4202 . This bacteriocin could be used to control B. cereus, which can cause diarrhoea and vomiting. Bt-BLIS: The B. thuringiensis BLIS include morricin 269, kurstacin 287, kenyacin 404, entomocin 420 and tolworthcin 524, produced by B. thuringiensis strains isolated in Mexico. These BLIS have been shown effective against Gram-positive and Gram-negative bacteria, including a etiologic agents of human diseases such as throat infections, scarlet fever, septicaemia, pneumonia, urinary tract infections, emetic and gastrointestinal syndromes and different types of fungal infections. Furthermore, activity of the five BLIS mentioned earlier was tested against isolates of S. aureus associated with bovine mastitis. All the S. aureus isolates were shown to be susceptible to the five bacteriocins synthesized by B. thuringiensis, mainly to morricin 269 and kurstacin 287, followed by kenyacin 404, entomocin 420 and tolworthcin 524 .
In this report, we propose a classification of Bt based on molecular weight, and functionality in single peptide and two-component peptide bacteriocins.
Because of the broad-spectrum nature of some B. thuringiensis bacteriocins, some may be included in antimicrobial strategies against virus, fungi, parasitic diseases and cancer, as well as in the immune system modulation . The potential applications of these peptides reinforce the importance of studying their biological properties.
As previously mentioned, nisin, a bacteriocin approved by the Food and Drug Administration, is employed to inhibit bacterial growth in processed food. Although the potential application can be quite diverse and requires greater knowledge and tests that may be used in different health sectors as pathogen control in hospitals, environment and new antibiotics for controlling human and animal diseases.
For decades, B. thuringiensis has been one of the best options, as it has been safely employed as a bioinsecticide and applied in different human consumption crops without showing adverse effects. In particular, thuricin CD is a good example as it displays specific activity against C. difficile and it has no effect on the good flora living in the intestine, as is the case with most traditional antibiotics .
E.L.S.M. was a recipient of a fellowship from the Consejo Nacional de Ciencia y Tecnología (CONACyT) México. M.A.R.L. is SNI member. The authors thank Secretaría de Investigación y Posgrado, Instituto Politécnico Nacional; Grant: 20151437.
Conflicts of interest
There are no conflicts of interest.
1. Carlet J, Jarlier V, Harbarth S, Voss A, Goossens H, Pittet D. Ready for a world without antibiotics? The pensières antibiotic resistance call to action. Antimic Resist Infect Contr
3. Dobson A, Cotter PD, Ross RP, Hill C. Bacteriocin production: a probiotic trait? Appl Environ Microbiol
4. Fischbach MA, Walsh ChT. Antibiotics for emerging pathogens. Science
5. López JE, Ochoa A, Santoyo G, Anaya JL, Medina E, Martínez M, et al. Bacteriocinas de bacterias Gram positivas: una fuente potencial de nuevos tratamientos Biomédicos. Revista Mexicana de Ciencias Farmacéuticas
6. Dobson A, Cotter PD, Ross RP, Hill C. Bacteriocin production: a probiotic trait? Appl Environ Microbiol
7. Zhoum Y, Choi YL, Sun M, Ziniu Y. Secondary metabolites of soil Bacillus spp. Microbiol Biotechnol
8. Klaenhammer TR. Genetics of bacteriocins
produced by lactic acid bacteria. FEMS Microbiol Rev
9. Liu C, Hu B, Liu Y, Chen S. Stimulation of nisin production from whey by a mixed culture of Lactococcus lactis and Saccharomyces cerevisiae. Appl Biochem Biotechnol
10. De la Fuente-Salcido N, Alanís-Guzmán MG, Bideshi DK, Salcedo-Hernández R, Bautista-Justo M, Barboza Corona JE. Enhanced synthesis and antimicrobial activities of bacteriocins
produced by Mexican strains of Bacillus thuringiensis. Arch Microbiol
11. Abriouel H, Franz Ch MAP, Omar NB, Galvez A. Diversity and applications of Bacillus bacteriocins
. FEMS Microbiol Rev
12. George RH, Symonds JM, Dimock F, Brown JD, Arubi Y, Shinagawa N. Identification of Clostridium difficile as a cause of pseudomembranous colitis. BMJ
13. Rea MC, Sit CS, Clayton E, O’Connor PM, Whittal RM, Vederas JC. Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile. PNAS
14. Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol
15. Favret ME, Yousten AA. Thuricin: the bacteriocin produced by Bacillus thuringiensis. J Invertebr Pathol
16. Barboza-Corona JE, Vázquez-Acosta H, Bideshi DK, Salcedo-Hernández R. Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis. Arch Microbiol
18. Guillet C, Join-Lambert O, Le Monnier A, Leclercq A, Mechaï F, France M, et al. Human listeriosis caused by Listeria ivanovii. Emer Infect Dis
19. Solórzano-Santos T, Miranda-Novales A. Resistencia de bacterias respiratorias y entéricas a antibióticos. Salud Pública de México
20. Stern NJ, Svetoch EA, Levchuk VP, Pokhilenko VD, Eruslanov BV, Mitsevich EV, et al. Inactivating Methicillin-Resistant Staphylococcus aureus and other pathogens by use of bacteriocins
OR-7 and E 50-52. J Clin Microbiol
21. Cherif A, Ouzari H, Daffonchio D, Cherif H, Ben Slama K, Hassen A, et al. A. Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1. 7, a new strain isolated from soil. Lett Appl Microbiol
22. Kamoun F, Mejdoub H, Aouissaoui H, Reinbolt J, Hammami A, Jaoua S. Purification, amino acid sequence and characterization of bacthuricin F4, a new bacteriocin produced by Bacillus thuringiensis. J Appl Microbiol
25. Arredondo JL, Villacaña RJ. Atlas bacteriológico. BAYER, Real Academia de Medicina, México
26. Cherif A, Chehimi S, Limem F, Hansen BM, Hendriksen NB, Daffonchio D, et al. Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis subsp. entomocidus HD9. J Appl Microbiol
27. Ugras S, Kazim S, Hatice K, Zihni D. Purification and characterization of the bacteriocin thuricin bn1 produced by Bacillus thuringiensis subsp. kurstaki Bn1 isolated from a Hazelnut Pest. J Microbiol Biotechnol
29. Chehimi S, Delalande F, Sable S, Hajlaoui MR, Van Dorsselaer A, Limam F, et al. Purification and partial amino acid sequence of thuricin S, a new anti-Listeria bacteriocin from Bacillus thuringiensis. Can J Microbiol
30. Chehimi S, Limam F, Lanneluc I, Delalande F, Van Dorsselaer A, Sablé S. Identification of three novel B. thuringiensis strains that produce the Thuricin S bacteriocin. Bt Res
31. Cherif A, Rezgui W, Raddadi N, Daffonchio D, Boudabous A. Characterization and partial purification of entomocin 110, a newly identified bacteriocin from Bacillus thuringiensis subsp. entomocidus HD110. Microbiol Res
32. Cleveland J, Montville TJ, Nes IF, Chikindas ML. Bacteriocins
: Safe, natural antimicrobials for food preservation. Int J Food Microbiol
33. Lee H, Churey JJ, Worobo RW. Biosynthesis and transcriptional analysis of thurincin H, a tandem repeated bacteriocin genetic locus, produced by Bacillus thuringiensis SF361. FEMS Microbiol Lett
34. Perrin M, Bremer M, Delamare C. A fatal case of Listeria innocua bacteremia. J Clin Microbiol
35. Kamoun F, Fguira IB, Hassen NBB, Mejdoub H, Lereclus D, Jaoua S. Purification and characterization of a new Bacillus thuringiensis bacteriocin active against Listeria monocytogenes, Bacillus cereus and Agrobacterium tumefaciens. J Appl Microbiol
36. Gray EJ, Lee KD, Souleimanov AM, Di Falco MR, Zhou X, Ly A, et al. A novel bacteriocin, thuricin 17, produced by plant growth promoting rhizobacteria strain Bacillus thuringiensis NEB17: isolation and classification. J Appl Microbiol
37. Lee KD, Gray EJ, Mabood F, Jung WJ, Charles T, Clark SR, et al. The class IId bacteriocin thuricin-17 increases plant growth. Planta
38. Honey bee diseases and pests: a practical guide. Agricultural and food engineering technical report. http://www.fao.org/3/a-a0849e.pdf
. [Accessed 20 December 2015]
39. Paik HD, Bae SS, Pan JG. Identification and partial characterization of tochicin, a bacteriocin produced by Bacillus thuringiensis subsp. tochigiensis. J Ind Microbiol Biotechnol
40. Ahern M, Verschueren S, Van Sinderen D. Isolation and characterization of a novel bacteriocin produced by Bacillus thuringiensis strain B439. FEMS Microbiol Lett
42. Rea MC, Alemayehu D, Ross RP, Hill C. Gut solutions to a gut problem: bacteriocins
, probiotics and bacteriophage for control of Clostridium difficile infection. J Med Microbiol