An approach to alternative strategies to control avian coccidiosis and necrotic enteritis

Morphology, Toxins, and Pathogenesis

C. perfringens is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium. It is commonly found as a normal inhabitant of the gastrointestinal tract (GIT) in humans and animals [3]. Distribution of C. perfringens is also common in environments such as soil, water, and feed [4]. It is an enteric pathogen that acquires nutrients from host animals by rapidly destroying tissues, and therefore, tissue necrosis is one of its pathological hallmarks. In recent years, there has been an increasing interest in understanding the pathogenesis and the prevention of NE in chickens in the absence of antibiotics. C. perfringens pathogenesis has been discussed extensively in a few studies such as Van Immerseel et al. and Prescott et al [5, 6]. However, this review will provide an update and progress of the NE together with the control attempts that are still under investigation. The C. perfringens produces at least 12 different toxins, which are associated with the occurrence of NE in chickens. Not all the C. perfringens inhabiting the chicken gut are pathogenic, and only few of the strains are virulent and are pathogenic. Four major extracellular toxin types, namely alpha (α), beta (β), epsilon (ε), and iota (ι), are produced by biotypes of C. perfringens A, B, C, D, and E [7]. Type A strains are most widespread and found in the GI tract as well as environment. All strains of C. perfringens produce α-toxin, whereas type A produces α-toxin as well as another pore-forming membrane-damaging β-toxin called NetB. NetB toxin has shown 38% identity to C. perfringens β-toxin and 31% identity to Staphylococcus aureus α-toxin. NetB toxin is responsible for necrotizing tissues causing perforations in the cell membranes of epithelial cells thus causing destruction and resulting in leakage of the intestinal contents [8]. The virulence of NetB toxin as well as the relation between the other different toxins has been studied in potential vaccine applications [7]. The detail of the NE antigen and the role in vaccine development as an alternative to antibiotics is discussed further in our review article.

NetB is thought to cause an initiation to the pathogenesis of NE and has been already identified in previous studies. However, there is a complex system and process involved in its complete pathogenesis and effect. The pathogenesis begins with the site colonization, multiplication, nutrients acquisition, and suppression of host innate and acquired immunity and therefore causing damage to the intestinal epithelial cells [6]. Colonization initiates when the mucosal barrier, containing antibacterial proteins such as mucin glycoproteins of the susceptible tissue is destroyed. The initial stage of the pathogenesis has also been found to involve the release of proteolytic enzymes causing destruction of villi and causing disruption of lamina matrix of the enterocytes [5, 9]. Lamina propria of intestine is infiltrated with inflammatory cells leading to an extensive disorder of intestinal integrity [9]. Such morphological changes in the intestinal tissues give an indication of the initiation of the pathology as shown by histopathology. A study has also reported that techniques such as real-time PCR have identified the mucosal gene such as MUC that is expressed in intestine [10]. The host defense mechanism against NE infection has been identified after identifying the specific gene such as MUC that is a step to understanding the NE model and mucin synthesis. Another advancement is an identification of a zinc metallopeptidase protein named NELoc-1 which might be an indication of proteolytic and collagenolytic enzyme release and can be also used in vaccine development [6]. Some of the advancement in the NE-specific loci has been reported after genome sequencing that carries NetB toxin causing NE disease in birds. However, in a study by Lee et al. [11], it has been stated that the presence of NetB toxin as detected by PCR does not guarantee the actual pathogenesis of NE. Therefore, this topic is still under investigation and needs more research to understand the complete effect of C. perfringens.

C. perfringens is a naturally occurring primary cause of NE in poultry. However, the severity of the disease depends upon various predisposing factors that eventually exacerbate the disease [12]. Some of the predisposing factors of the disease include coccidiosis, dietary factors, management stress-causing immunosuppression, and an overall imbalance of commensal microbiota [13]. Coccidiosis infection and its prevention, dietary factors such as high crude protein (CP), high fiber, and nonstarch polysaccharides (NSP), as well as management factors such as cold and heat stress, stocking density, that contribute to NE will be discussed further in the review (Figure 1) along with dietary intervention to combat NE in poultry.

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Figure 1. Predisposing factors for necrotic enteritis (NE) in chickens.

Coccidiosis

One of the common predisposing factors of NE is a coccidiosis infection [7]. Chicken coccidiosis is considered an infectious protozoan disease caused by the genus Eimeria, under which common species include acervulina, maxima, tenella, necatrix, mavati, mitis, praecox, and brunetti [14]. Eimeria-assisted introduction, especially Eimeria maxima, has been found to be a major risk factor to promote C. perfringens strain. Such association has been related to cause the epithelial surface damage, serum release, and mucogenesis induction, which overall provides a niche for C. perfringens to colonize and proliferate. Such species have been identified extensively in different parts of the small intestine and ceca of chickens. The major pathogenesis behind any Eimeria species is because of rapid multiplication within the mucosal epithelia of the intestinal lining that results in inflammation, disruption of gut integrity, and hemorrhage leading to secondary infections, morbidity, and mortality [15]. Coccidiosis infection causes rapid proliferation of sporozoites within the lining of the intestine, which is disruptive, causing damage to the epithelial surface, which leads to hemorrhages, reddish, orange, or pink viscous exudate, and excess mucin production. The intestinal damage caused by coccidia is an essential predisposing factor for NE resulting in overgrowth of C. perfringens and toxin production [15]. This leads to leakage of serum into the gut, thus stimulating mucus production and providing a richer source of nutrients for C. perfringens proliferation [10]. In addition, direct invasion of Eimeria compromises the barrier function and the immune system of birds. Host mucogenic response to a coccidial infection and the intestinal damage provides an advantage for the onset of NE [10]. A study by Collier et al. [15] observed an increase in the expression of interferon (IFN)-γ, interleukin (IL)-10, and IL-4 in the chicken intestine at the early phase of combined coccidiosis and NE infection. Infection of Eimeria has shown an increase in the intestinal mucin RNA expression, goblet cell number, and theca size, which indicates that there is a host intestinal mucogenic response [13]. An increase in the regulation of the mucin gene such as MUC2 expression during coccidiosis infection has further acted as a medium for C. perfringens growth [15].

Chemicals, vaccines, and ionophores alone or in combination have been used as effective strategies to control coccidiosis for extended periods of time. Owing to drug-resistance and the ban of some coccidiostats and ionophores in poultry production, alternative control strategies have included dietary interventions, which have become an area of focus for the poultry industry recently [16]. Dietary intervention, mainly involving natural products or plant products, has shown some success with the control of Eimeria in chickens. Identification of dietary interventions that can combat such gut health dysbiosis created by coccidiosis is a major challenge that will be faced by the current and future poultry industry [16]. However, while attempting to control Eimeria in chickens, NE is also being studied because both of these diseases complement each other in host–pathogenic response and decrease performance. Therefore, a complete understanding of the predisposing factors and relationship between NE and coccidiosis and the mitigation strategies through various intervention strategies is necessary to control and/or prevent both diseases from occurring in poultry. Dietary interventions that have been used against coccidiosis are described below:

Interventions Against Coccidiosis

Management Factors

Stress including environmental stressors such as heat or cold stress, management such as vaccination, ventilation, and stocking density, as well as other diseases, play a role in NE occurrence [68, 69, 70, 71]. Cold stress has been associated with lesion production in Clostridium challenged broilers chickens [68]. Tsiouris et al. [68] reported significant increases in the cecal C. perfringens counts and severe NE lesions when birds were exposed to cold stress. When birds are exposed to low temperatures, immunosuppression occurs, which affects both humoral as well as cellular immunity [70]. When birds are under cold stress, feed intake will increase to compensate for heat loss. This will also bring a change in internal energy production, predominantly by producing reactive oxidative species [71]. During cold months, ventilation in the house is less used thus resulting to increase in ammonia levels and moisture in litter that can proliferate cocci and NE in the litter. Heat stress is another most important environmental stressors in poultry production [72]. The release of serum corticosterone, as well as heat shock proteins in long-term heat-stressed chickens, have been shown to result in greater number of C. perfringens. The authors also reported that heat stress lowers the bacterial counts of Lactobacillus and Bifidobacterium spp. but increases the counts of coliform and Clostridium spp. [72]. Heat stress has been linked to impaired intestinal morphology following pathogenic bacteria invasion through the intestinal epithelium [72, 73]. A study with long-term heat stress to broiler chickens reported stress-induced regulation of intestinal inflammation, particularly a stress-induced decrease in the intestinal polymorphonuclear cell infiltration [72]. This inflammatory process leads to necrotic foci in the intestinal mucosa, which increases the intestinal goblet cells. Alterations in the intestinal epithelium integrity could disturb the microbiota homeostasis leading to enteritis [73]. Stress, heat, or cold can also cause inflammation of the intestine that leads to a change in the immune status of the gut which can induce proliferation of C. perfringens. These compromises both humoral and cellular immunity, leading to a greater number of C. perfringens being shed onto the litter thus impacting the intestinal microbiota and overall performance of chickens.

Stocking density is another management factor and has an important role in producing NE. The high stocking density of chickens has been found to be associated with a high number of C. perfringens in the ceca as demonstrated by higher gross lesion score in ceca [74]. The mechanism related to the stocking density and immunity of birds has been described as the humoral immune system being compromised that affects the bird’s ability to produce antibiotics and combat the infection [74]. A higher density is also associated with an increased level of nitrogen and moisture in the litter thus downgrading litter quality and increasing bacterial and coccidial oocyst counts in the litter. The bacterial composition of the poultry litter has been shown to produce a change in chicken gut microbiota. The predisposing effect of high stocking density on NE is one of the management factors, and further studies might contribute to considerate the detail mechanisms of this factor.

Viral infections and vaccines used against diseases like Marek’s disease, chicken infectious anemia, and infectious bursal disease have been shown to cause immunosuppression, leading to a greater turnover in the intestinal immune system, which can predispose broilers to NE disease [75]. Also, the infectious bursal disease vaccine has proven to be a predisposing factor to induce NE, where the vaccine was either supplemented at the intermediate dose or 10 times higher the recommended dose [75]. The bursa of Fabricius, an organ associated with humoral immunity, when targeted by a viral disease such as infectious bursal disease, caused immunosuppression in broilers, which led to the secondary infection such as Escherichia coli and C. perfringens [75]. The virus affects the lymphoid cells targeting bursa and suppressing humoral immunity. Coccidiosis also causes immunosuppression leading to gastrointestinal imbalance, which gives rise to C. perfringens infection. The details have been described in a separate section above.

Certain breeds of broilers have been found to be severely affected by C. perfringens, causing NE. Some major commercial broiler breeds such as Cobb, Ross, and Hubbard when orally infected with the bacteria and E. maxima developed the disease more rapidly compared to the rest [76]. The research mentioned above reported that the antibody levels of NetB toxins were greater in Cobb chickens, which means Cobb chickens are more susceptible to NE compared with the Ross or Hubbard chickens [76]. Disease susceptibility was measured in the form of body weight loss, intestinal lesions, and serum antibody levels against NetB like toxin. Cobb chickens had a greater loss in body weight and increased the intestinal lesion scores after coinfection with E. maxima and C. perfringens. It has been presumed that the attribution toward the higher susceptibility in some of the broiler breeds against NE could be because of the difference in immune-related genes in such breeds.

Enzymes

Supplementation of NSP degrading enzymes and their role in ameliorating NE has been studied in poultry. Multicarbohydrase blends such as xylanase, amylase, alpha-galactosidase, pectinase, and β-glucanase have shown to reduce the deleterious effects of NSP, reducing intestinal digesta viscosity, improving the nutrient digestibility of diets, and ultimately reducing dietary stress on the gut [77, 78, 79, 80]. Diets that have a higher proportion of NSPs, particularly based on wheat and have been supplemented with exogenous enzymes such as xylanase, have shown lower digesta viscosity thus lowering coccidiosis and the incidence of NE [80, 81].

Studies by Van Immerseel et al. [82] and Jackson et al. [83] have reported that xylanase has been successful in improving animal performance resulting in reduced digesta viscosity and fermentation, which eventually suppressed C. perfringens in the small intestine. Such enzymes have also shown an improvement in the ileal digestibility of CP and amino acids in broilers [84]. Jia et al. [85] reported that the supplementation of multicarbohydrase enzymes in birds fed high dietary levels of flaxseed resulted in a reduction of intestinal digesta viscosity because of increased water-binding capacity of complex carbohydrates, thus reducing C. perfringens numbers in the intestine. Also, feeding a direct-fed microbial, cellulase, and xylanase that were produced in the gut resulted in a reduction in both viscosity and C. perfringens proliferation compared with the control diets [86]. Xylanases have also shown a prebiotic-like effect in poultry. It has been found that enzymes hydrolysis produces galacto-oligosaccharide, gluco-oligosaccharide, and manno-oligosaccharides in the chicken GIT and supports the growth of Bifidobacterium and Lactobacillus. Inhibition of pathogenic species such as C. perfringens can occur after the growth of lactic acid bacteria in the lower GIT because of the exogenous enzyme hydrolysis [86]. The mechanism can be explained as a competitive exclusion that prevents pathogens such as C. perfringens from binding to the epithelium, which reduces colonization and allows for better digestion and nutrient absorption within the gut [65]. Xylanase supplementation in the wheat-based diet has shown to enhance dietary utilization of nutrients in C. perfringens challenged broilers by ameliorating the retarded growth, improving the feed conversion ratio, and improving intestinal barrier function [60, 87]. In another study by Jackson et al. [83] addition of B-mannanase to the corn-based diet improved performance and reduced lesion scores in birds challenged with both Eimeria spp. and C. perfringens. However, it should be noted that for the effectiveness of enzymes, the appropriate substrate must be available to act upon, such as wheat vs. corn, and the water-soluble portion of the ingredients, which aids in the enzyme hydrolysis pattern better [60]. Moreover, the enzyme is used as energy sparing additive in the feed rather than a cocci prevention tool so producer should not only rely on the enzyme to do the function of reducing cocci but as a combination of tools to combat the disease when no antibiotics are used.

Phytochemicals and Essential Oils

Phytochemicals have been used as antiviral, antimicrobial, antioxidant, and anti-inflammatory in human health [88]. The mechanism of action of phytochemicals is claimed to be their ability to disintegrate bacterial cell membranes and penetrate the bacterial cells. The antibacterial mechanism of phytochemicals is associated with killing the pathogenic bacteria directly and partitioning the bacteria into their lipid contents and mitochondria. The lipid interaction interferes with bacterial metabolism, the cell wall, and membrane permeability, leading to extensive leakage of critical molecules and ions from the bacterial cells of such harmful bacteria [89]. The beneficial effects of EO may be related to a direct antimicrobial effect on bacterial cells and an indirect effect by modulating gut microbiota and digestive functions [90]. Dietary phytochemicals have shown to induce the enzyme expression in cellular antioxidant response. A study that used magnolia bark extract in a combined challenge of E. maxima and C. perfringens reported in the upregulation of enzymes such as aflatoxin B1 aldehyde reductase, catalase, and superoxide dismutase [91]. The main bioactive compound in the phytochemicals are polyphenols and have been used as a potential antibiotic alternative in swine and poultry [91].

Some of the common phytochemicals and EO are herbs and spices including thyme, oregano, rosemary, coriander, cinnamon, and green tea have shown a positive influence in poultry [92, 93]. Supplementation of a mixture of capsicum and Curcuma longa oleoresins have shown better performances by increasing body weight and reduced gut lesion scores and improved the immunity by reducing mRNA expression of IL-8 in NE-infected broilers compared with infected and nonsupplemented group [92]. The different portions of EO blends produce variations in the effectiveness when added to poultry diets. Some of the EO containing thyme and clove are also capable of increasing body weight and improving growth performance while decreasing C. perfringens count in the jejunum, especially during the first half of the growing period [93]. A uniform blend of EO containing thymol, carvacrol, eugenol, curcumin, and piperin has shown a reduction in the colonization and proliferation of C. perfringens in the gut of broiler chickens [94]. When used as a preventive measure, thyme and clove continuously supplemented in feed, demonstrated a beneficial effect against C. perfringens by lowering the bacterial counts and alleviating intestinal damage.

The antibacterial effect of EO, along with stimulated digestive enzymes seem to have stabilized the intestinal bacterial microflora, thus inactivating C. perfringens toxins. When thymol was used as a preventative compound, a reduction in C. perfringens proliferation in the broiler intestine as well as in fecal excretion was observed [93, 94]. In an in vitro study, carvacrol supplementation reduced C. perfringens by 2 to 3 log and when mixed with chicken ileal contents later, results were found to be similar. However, the role of phytochemicals and EO to define the molecular and cellular mode of action to control NE needs to be studied further.

Organic Acids

Organic acids, both SCFA and medium-chain fatty acids, have proven to be effective against NE infections. Several in vitro and in vivo experiments have shown beneficial effects when organic acids have been used individually or as a blend in chickens. Although antibacterial mechanisms of these organic acids are poorly understood, they are known to have a bacteriostatic or bactericidal effect whenever possible. A reduction in the intracellular pH via the entry of undissociated acids into the bacterial cell and subsequent dissociation in the cytoplasm is believed to be the major mechanism of organic acids. Butyric acid has shown an increased expression of intestinal tight junction thus decreasing intestinal permeability in broiler chickens [95]. Lactic acid bacteria that ferment carbohydrates present in feed produce one of the organic acid, lactic acid, which lowers the pH of the surrounding environment and inhibits the growth of specific pathogens such as E. coli, Salmonella typhimurium, and C. perfringens [96].

Blends of acetic, butyric, and lactic acids have resulted in weight gain and feed conversion ratio benefits with elevated CD3+ T-cells in the ileum of 7 D of age broilers challenged with C. perfringens [97]. Similarly, blends of formic, acetic, propionic, sorbic, caprylic, and capric acids have resulted in low clostridial loads and improved feed conversion ratio in broilers [98]. Change in pH being their primary mode of action and the combined effects of different organic acid compounds have been able to combat such effect against C. perfringens. However, such various blend did not improve growth performance or reduced mortality [98] and the reason behind this has not been explained. In a study by Geier et al. [98], organic acids resulted in an increase in the number of lactic acids producing bacteria in the intestine and had an increased number of T-cells in the intestinal mucosa of broilers. Supplementation of 0.2% encapsulated organic acids to the diet demonstrated an improvement in the proliferation of Lactobacillus spp. and diminished the population of harmful bacteria including C. perfringens in poultry gut contents [99, 100]. It has been claimed that the positive effect of organic acids can be attributed to the trophic effect of organic acid products on the intestinal mucosa and immune cells. The direct effects of organic acids on specific Clostridium strains as well as Clostridium loads in the intestine require further exploration.

Probiotics and Prebiotics

Probiotics are becoming a common intervention method used for NE prevention and control in the postantibiotic era. Some of the commonly used probiotic bacteria used in broilers infected with NE are Lactobacillus johnsonii, Bacillus licheniformis, Bacillus amyloliquefaciens, and B. subtilis [101, 102, 103, 104, 105, 106]. Such probiotic bacteria have been shown to improve both growth performance and intestinal immunity. Probiotics can directly compete against the pathogenic bacteria for nutrition and adhesions, which then produce metabolites that can directly inhibit the growth of pathogenic bacteria. For example, probiotic bacteria such as Bacillus subtilis have spores, which have proven to be highly resistant against high heat and low bile pH in the GIT, thus providing a protective mechanism to the chicken.

The beneficial effects of probiotic bacteria, mostly because of the interaction of such probiotic bacteria with the diversity of bacteria in the gut has also been observed. A study by Lin et al. [104] investigated the population of microbiota in broiler ileum samples that had been challenged with C. perfringens. They reported an imbalance in the ileum microbiota of Clostridium challenge birds. It was also demonstrated that the microbiota was returned to normal after feeding B. licheniformis, with enhanced mucosal immune activity and improved epithelial barrier function [105]. Bacillus licheniformis has also shown to reduce the dysbiosis of the cecal microbiome which can occur because of a challenge with C. perfringens [105]. A study by Zhou et al. [106] on the effect of B. licheniformis on broilers challenged with C. perfringens-induced NE, reported an increase in the expression of lipid metabolism-related genes and reduced antioxidant stress. When B. licheniformis was added to the diet at 8 × 10⁷ CFU/g of feed, the results showed a similar effect to birds provided virginiamycin where a reduction in lesion score severity and NE mortality was observed [106]. On the other hand, Lactobacillus-based probiotics have been successful in mitigating some of the health issues associated with birds challenged by C. perfringens. Supplementation of Lactobacillus acidophilus and Lactobacillus casei have been found to lower the cecal counts of C. perfringens in the diet compared to birds fed a control diet [106].

L. johnsonii based probiotics have shown enhanced intestinal development and a balanced microbiota within the intestine of treated birds compared with the C. perfringens-challenged control birds [107]. Their study also found that L. johnsonii significantly increased the abundance of all Lactobacillus spp., and decreased Streptococcus spp., as well as Enterobacteriaceae in the jejunum and ileum. An increase in the lipolytic genes such as PPARα and CPT-1 in the study suggested that the addition of L. johnsonii may have improved the suppression of fatty acid oxidation in the subclinical NE group chickens [107]. Lactobacillus fermentum–based probiotic supplementation in NE-infected birds showed downregulation of Toll-like receptor-2 and IFN-γ and upregulated the expression of IL-10 [108]. These data show that L. fermentum can maintain intestinal homeostasis by balancing between proinflammatory and anti-inflammatory cytokines.

Prebiotics prevent the colonization of bacterial pathogens in the GIT, lower the gut pH through SCFA production and thus stimulate the immune system. Arabinoxylans are polysaccharides that have been found in the cell wall of cereal-based grains such as rice and wheat. Arabinoxylo-oligosaccharides (AXOS) are derived by partial hydrolysis of arabinoxylan, and they have shown a beneficial effect against pathogenic bacteria like Salmonella because of the ability to decrease bacterial shedding in the feces [109]. With regards to NE control and prevention, AXOS also have shown an improvement over feed efficiency as well as increased the quality of SCFAs in broilers [75]. Selective stimulation of beneficial bacteria, suppression of pathogenic bacteria, optimizing colon function, and stimulating immune functions are some of the functions of AXOS inside the gut. However, the effects of AXOS inclusion on gut microflora, gut physiology, and digesta passage rate have not been explored in broilers.

Yeast cell wall (YCW) is another type of prebiotic that has been supplemented in NE-infected chickens individually or as a combination [110, 111]. Yeast cell walls contain protein, mannans, and ß-glucans. A study by M’sadeq et al. [111] reported that yeast cell wall extract from the YCW of Saccharomyces cerevisiae has potential to act as an alternative to salinomycin or zinc bacitracin, which provided positive effects by improving feed intake, weight gain, and livability in C. perfringens–challenged broilers. A combination of YCW and Bacillus has shown an improvement in intestinal health by lowering the serum endotoxin concentration of C. perfringens and increasing IgA levels in the ileal mucosa of broilers [110]. Such a combination has also lowered the bacterial count of C. perfringens and E. coli while increasing B. subtilis in the cecum of broilers. However, the study did not find an improvement in intestinal lesion scores and ileal morphology. A possible explanation behind this might be because of a lack of intestinal turnover and regeneration of the intestinal epithelium. However, for the better control of NE, specific probiotic strains should be explored with their particular mode of action. Also, for the prebiotics to work better in the gut, the specific probiotic bacteria that they stimulate and act upon specific Clostridium strains need to be explored further.

Bacteriophages

Bacteriophages (BP) are viruses that infect or replicate within the bacteria, leading to an attachment to specific hosts and kill them by lysis and cell death. Bacteriophages against C. perfringens have been sequenced from poultry offal, feces, and production run-off which have specific anti-Clostridium activity [112]. Such BP, used either as a single sequence or a cocktail of sequences, have been utilized to mitigate C. perfringens infection in the field as well as in research trials with broilers. In a study by Miller et al. [113], different BPs were characterized according to their morphological and phenotypic characteristics and used against E. maxima (5,000 oocysts) and C. perfringens challenge in broilers. Oral gavage of Eimeria was performed at day 14 followed by C. perfringens challenge (10⁸ cfu/mL) on days 19, 20, and 21. Following the pathogen challenge, BP-derived toxoid vaccine preparation containing cocktails of different BP (INT-401) was used either via drinking water, oral gavage, or via feed (10⁵ PFU/mL). These results indicated that there is a potential benefit to a BP cocktail, administered via drinking water or feed to control NE in broilers raised in floor pen environment. In another study by the same authors, oral administration of BP cocktail showed a significant decrease in mortality during day 0 to 42 of an experiment together with improvements in weight gain, FCR, and total mortality [114, 115]. Phages enzymes such as endolysins and mucolytic enzymes, including murein hydrolase, have shown to combat infections that are induced by C. perfringens. In some of the cases, BP and such phage enzymes have shown to have a lytic effect against Clostridium infection by directly binding to the peptidoglycans of the cell walls of gram-positive bacteria. Until now, the lysis mechanism of BP is only one mechanism among various mechanisms, and other mechanisms still need to be studied. Lysogenic properties of BP fail to produce an effect against bacteria as it can switch to the nonlysing phase. Therefore, few more studies need to be performed to identify the role of BP against the pathogens. Also, for the effectiveness of a BP, high specificity against a particular bacterium, allowing its ability to infect the bacteria needs to be explored.