Innovative Approaches Against Planktonic and Biofilm-Producing Clostridium perfringens Associated with Necrotic Enteritis

Simple Summary

Necrotic enteritis (NE) is an important bacterial disease affecting poultry and causes major economic losses worldwide. It is caused by Clostridium perfringens (C. perfringens), an aerotolerant anaerobic bacterium that produces many harmful toxins, most notably NetB and alpha-toxin, and forms protective biofilms that help it survive in poultry environments and resist antimicrobial treatments. The steady emergence of antimicrobial-resistant strains has made controlling this disease increasingly challenging. This review summarizes the epidemiology and pathogenicity of C. perfringens associated with NE, and discusses promising antimicrobial-free approaches for its control. Particular attention is given to strategies targeting both planktonic and biofilm-forming cells, including their effectiveness under in vitro, in vivo, and field conditions. The review highlights the need for sustainable and effective alternatives to conventional antimicrobials to control NE, reduce antimicrobial resistance, and improve poultry health and production.

Abstract

Necrotic enteritis (NE), caused by the Gram-positive, anaerobic bacterium Clostridium perfringens (C. perfringens; CP), is one of the most economically important diseases affecting the poultry industry worldwide. The pathogenicity of C. perfringens is attributed to the production of more than twenty extracellular toxins and enzymes, with strains classified into seven toxinotypes (A–G) based on their major toxin genes. While the alpha-toxin gene (cpa) is present across all C. perfringens toxinotypes, netB is recognized as the primary virulence determinant of avian NE. However, toxin-gene carriage alone does not establish toxin expression or disease causation. Cpb2 and tpeL have also been associated with virulence in some C. perfringens isolates, although their independent contributions to NE pathogenesis remain less firmly established. The increasing prevalence of antimicrobial-resistant C. perfringens strains, including multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) isolates, together with the organism’s ability to form biofilms, has complicated disease prevention and treatment by enhancing environmental persistence and tolerance to antimicrobial agents. Distinct from reviews that primarily address NE pathogenesis or antibiotic alternatives as broad terms, this review integrates antimicrobial resistance with planktonic and biofilm-associated C. perfringens. Furthermore, it critically evaluates alternative interventions based on their activity against these bacterial states and the strength of supporting in vitro, in vivo, and field evidence. In conclusion, this review highlights the urgent need for sustainable, antibiotic-free strategies to combat NE. Advancing the development and field validation of these alternative approaches may contribute to reducing antimicrobial resistance, limiting biofilm-associated persistence, and improving poultry health and productivity.

1. Introduction

Clostridium perfringens (C. perfringens, CP) is a Gram-positive, anaerobic, spore-forming bacterium that is a natural component of the poultry gut microbiota [1]. The presence of approximately twenty different extracellular enzymes and toxins is the basis for C. perfringens pathogenicity [2]. Under the expanded toxinotyping scheme, C. perfringens strains are classified into seven toxinotypes (A-G) according to the presence of the major toxin genes cpa, cpb, etx, iap/ibp, cpe, and netB [3,4]. α-toxin is produced by all seven types [5]. The enteritis agent alpha-toxin is characterized by its enzymatic activity and host-cell signaling features. Lately, C. perfringens type A has been described as the major type associated with enteritis in chickens [6,7] (Figure 1).

Figure 1.

Pathogenesis, economic impact, and alternative control strategies for C. perfringens-induced necrotic enteritis (NE) in poultry. C. perfringens induces NE through extracellular toxins, leading to intestinal damage and significant annual economic losses of $6 billion USD. Excessive bacterial proliferation (up to ${10}^9$ CFU/g) is triggered by confirmed predisposing factors (Coccidia, mycotoxins, immunosuppression). Given increasing concerns regarding antimicrobial resistance and biofilm-associated persistence, integrated control strategies involving probiotics, phytogenics, and emerging nanotechnologies are being investigated as potential approaches to maintain gut health and reduce NE-associated losses.

Necrotic enteritis (NE) is a bacterial disease caused by strains of C. perfringens [1], particularly types A, C, and G [8]. The disease is found in ducks, wild geese, Laysan albatrosses, mute swans, bobwhite quails, wild crows [9], and turkeys [10]. The disease is brought on by an excessive rise in the number of C. perfringens bacteria in the gastrointestinal tract (GIT) in combination with risk factors such as exposure to mycotoxins, diets high in non-starch polysaccharide grains, coccidia infection, and immunosuppressive viruses such as those that cause Gumboro and Marek’s disease, and chick anemia; all lead to decrease the animals’ resistance to GIT infections, causing severe NE [11,12]. The C. perfringens population in a healthy bird is 10²–10⁴ CFU/g digesta; however, during a disease, this number will rise to 10⁷–10⁹ CFU/g digesta [13] (Figure 1).

The clinical form is characterized by acute symptoms, including depression, ruffled feathers, diarrhea, dehydration, and a high feed conversion ratio (FCR). This form is severe and can result in daily mortality rates of up to 1% and a total of 10–40% deaths [14]. The most prevalent type of NE in the field is subclinical, which causes a decrease in weight gain and an increase in FCR, which ultimately results in poor performance and economic losses [14,15]. NE imposes a substantial economic burden on poultry production through mortality, reduced body-weight gain, impaired feed conversion, treatment and prevention costs, carcass losses, and delayed attainment of market weight. The global economic burden has been widely estimated at approximately USD 6 billion annually, although this figure represents an industry-level estimate rather than a harmonized global cost-of-disease analysis. Regional studies further demonstrate that the economic consequences of NE vary according to disease severity, production system, market conditions, and whether clinical or subclinical disease predominates. In particular, subclinical NE may generate substantial losses through persistent reductions in growth and efficiency despite relatively low mortality. Because directly comparable national monetary estimates remain limited, both economic metrics and experimentally quantified production losses are summarized in Table 1 to illustrate the global production burden of NE [14,15,16,17,18,19,20].

Table 1.

Reported economic and production impacts of necrotic enteritis across poultry-producing regions.

Region	Study Setting	Economic Parameters	Reported Effect	Evidence Type	Limitation	Reference
Global	Commercial poultry industry	Estimated annual economic burden	Approximately USD 6 billion annually due to production losses and disease-control costs	Global industry estimate/literature synthesis	These estimates are not based on a standardized multinational cost-of-illness analysis, as regional production costs and disease prevalence vary.	[16,17]
United States	Economic model of SNE using hypothetical 20,000-bird broiler flocks	Body weight, FCR, producer loss and additional feed cost	SNE modeling reduced body weight by 12% and increased FCR by 10.9%, driving estimated flock losses of USD 878.19–1480.52 alongside USD 370.49–739.38 in extra feed costs.	Economic modeling study	Rely on historical costs and an assumed 20% SNE incidence, meaning they cannot be directly applied to modern production systems.	[18]
Canada	Controlled broiler NE model	Mortality and intestinal lesions	Variant IBDV pre-exposure combined with C. perfringens challenge triggered 10–15% mortality and caused histopathological NE lesions in up to 70% of birds.	Experimental in vivo study	Controlled challenge model rather than commercial field-economic assessment	[14]
Australia	Controlled subclinical NE challenge in broilers	BWG and FCR	At day 16, NE challenge reduced BWG by 27% and worsened FCR by 11 points; by day 35, BWG remained 12% lower and FCR 5 points poorer than unchallenged controls	Experimental in vivo study	Focuses on performance loss rather than financial cost, while experimental challenge severity may not match all field outbreaks.	[19]
Egypt	Experimental C. perfringens infection in Cobb and Arbor Acres broilers	Growth, mortality, total return and economic efficiency	Untreated challenged birds had lower growth and returns than treated ones; Cobb birds yielded LE 39.02 when infected versus LE 44.91 when antibiotic-treated.	Experimental economic assessment	Reflects local, study-specific trial costs rather than nationwide economic burden.	[20]
China	Experimental C. perfringens challenge in broilers	Growth performance and intestinal injury	C. perfringens challenge significantly impaired growth performance together with intestinal barrier and inflammatory outcomes; dietary nisin mitigated these effects	Experimental in vivo study	Evaluates intervention efficacy rather than regional or national financial burden.	[15]

NE, necrotic enteritis; BWG, body-weight gain; FCR, food conversion rate; SNE, subclinical necrotic enteritis; IBDV, Infectious Bursal Disease Virus.

As illustrated in Table 1, geographic designation refers to the setting or scope of the cited study and should not be interpreted as a regional prevalence estimate. Monetary estimates, experimental production losses, and controlled challenge outcomes are presented separately because differences in production systems, challenge models, disease prevalence, market prices, and economic assumptions preclude direct quantitative comparison across regions.

Several reviews have addressed the pathogenesis, predisposing factors, host responses, and prevention of C. perfringens-associated NE, while others have focused on specific antibiotic alternatives, including probiotics, phytogenics, organic acids, and other feed additives [1,8,21,22]. More recent work has also highlighted the challenges associated with translating experimental NE-control strategies into commercial poultry production [23]. The present review extends these perspectives by integrating toxin-mediated virulence and antimicrobial resistance with the distinct biology of planktonic and biofilm-associated C. perfringens. Particular emphasis is placed on differentiating antimicrobial resistance from biofilm-associated tolerance and persistence and on critically evaluating non-antibiotic interventions according to the level of evidence available, including activity against planktonic cells, prevention of biofilm formation, effects on established biofilms, efficacy in experimental poultry models, and evidence from field applications. By comparatively examining probiotics, prebiotics, synbiotics, organic acids, bacteriophages, antimicrobial peptides, phytogenics, nanoparticles, and green nanotechnology within this framework, this review aims to identify current evidence gaps and the translational requirements for sustainable control of NE in poultry.

Literature Search Strategy

This narrative review was informed by a structured literature search of PubMed/MEDLINE, Scopus, and Web of Science Core Collection from database inception through August 2026. Search terms were used individually and in Boolean combinations and included “C. perfringens,” “necrotic enteritis,” “poultry,” “broiler,” “toxinotype,” “NetB,” “CPB2,” “TpeL,” “alpha toxin,” “virulence,” “pathogenesis,” “quorum sensing,” “antimicrobial resistance,” “biofilm,” “persistence,” “probiotics,” “prebiotics,” “synbiotics,” “organic acids,” “phytogenics,” “essential oils,” “antimicrobial peptides,” “bacteriocins,” “bacteriophages,” “nanoparticles,” and “green nanotechnology.” Peer-reviewed original studies and relevant reviews addressing C. perfringens virulence, antimicrobial resistance, biofilm biology, necrotic enteritis, and non-antibiotic interventions were considered. Priority was given to recent primary studies, particularly those published from 2022 to 2026, while earlier seminal studies were retained when required to establish foundational mechanisms. Studies involving other bacterial species were included only when they provided relevant mechanistic context and were clearly distinguished from evidence directly demonstrated in C. perfringens. Duplicate publications, conference abstracts without sufficient data, and studies lacking direct relevance to the scope of the review were excluded. Reference lists of relevant articles were also screened to identify additional eligible studies.

2. Taxonomy

Taxonomy of C. perfringens was determined adopting the National Center for Biotechnology Information as follows: Kingdom: Bacteria, Phylum: Firmicutes, Class: Clostridia, Order: Clostridiales, Family: Clostridiaceae, Genus: Clostridium, Species: Clostridium perfringens or Bacillus perfringens or Bacterium welchii [24].

3. C. perfringens Toxinotypes

The pathogenic potential of C. perfringens is associated with a diverse repertoire of toxins and extracellular enzymes. Under the expanded toxinotyping scheme, C. perfringens strains are classified into seven toxinotypes (A–G) according to the presence of genes encoding the major toxins: alpha toxin (cpa), beta toxin (cpb), epsilon toxin (etx), iota toxin (iap/ibp), enterotoxin (cpe), and NetB (netB) [3,4].

Specifically, toxinotype A carries cpa without the defining major toxin genes of types B–G; toxinotype B carries cpa, cpb, and etx; toxinotype C carries cpa and cpb; toxinotype D carries cpa and etx; toxinotype E carries cpa and the iota-toxin genes; toxinotype F carries cpa and cpe; and toxinotype G carries cpa and netB [3,25,26]. Thus, enterotoxin-producing strains classified by the presence of cpe together with cpa correspond to toxinotype F, whereas toxinotype E is defined by alpha- and iota-toxin genes [3,27,28,29,30].

Under this expanded scheme, NetB-positive strains are classified as toxinotype G. Some earlier poultry studies described these isolates as type A because they were published prior to the adoption of the A-G system; historical classifications are retained when reporting those studies but clarified using current terminology [3,4].

The genes encoding α-toxin, perfringolysin O, collagenases, sialidases, hyaluronidases, and collagen adhesion protein are located on the chromosome in C. perfringens type A/G strains. In contrast, key NE-associated toxins, namely NetB, Beta2 toxin, and C. perfringens large cytotoxin (TpeL), are encoded on plasmids [31] (Table 2).

Table 2.

Toxins and enzymes produced by C. perfringens in poultry.

Toxin Type	Synonym	Gene	Gene Location	Significant Effect	References
Alpha-toxin	Phospholipase C	cpa, plc	C	To hydrolyze cell membrane phospholipids	[3,27,28,29,30]
Perfringolysin O	θ-toxin (pore-forming toxin)	pfoA	C	Pore formation via binding to cholesterol-comprising cell membrane	[3,32,33,34]
Collagenase	κ-toxin	colA	C	To degrade collagen, which is the main component of the connective tissues of host cells	[3,31,35]
Sialidase	Secreted major neuraminidases	nanI	C	Involved in the removal of sialic acids from a variety of glycoconjugates on cell membranes	[24,36]
	Non-secreted neuraminidases	nanH	C
	Secreted neuraminidases	nanJ	C
Hyaluronidase	µ-toxin	nagH	C	To degrade hyaluronan coating cells, allowing direct contact between pathogen and host cells; or to degrade hyaluronan, leading to viscosity reduction, facilitating increased permeability of the connective tissues	[24,37]
		nagI	C
		nagJ	C
		nagK	C
		nagL	C
Collagen adhesion protein	Cell surface protein	cnaA	C	Binding of C. perfringens to collagen types IV, V and gelatine	[17,24]
NE B-like toxin	Pore-forming toxin	netB	P	To form heptameric, hydrophilic pores with a central diameter of approximately 26 Å	[22,38]
Beta2 toxin	Pore-forming toxin	cpb2	P	Pore-forming, leading to cell disruption	[22,39]
Toxin C. perfringens large cytotoxin	Large clostridial toxin	tpeL	P	Ras-specific glucosyl transferase activity, inactivating the Ras signaling pathway, leading to apoptosis	[22,40]

C, chromosome; P, plasmid; NE, necrotic enteritis.

When interpreting toxin profiles, the presence of a toxin gene should be distinguished from transcription, protein production, biological activity, and demonstrated contribution to disease. PCR-based detection establishes genetic potential but does not by itself demonstrate toxin expression or causality. Accordingly, associations between cpa, netB, cpb2, or tpeL and NE should be interpreted according to the level of supporting evidence, with greater weight given to toxin-expression studies, functional assays, and infection experiments using defined mutants and complemented strains.

The pathogenic mechanism of C. perfringens in poultry relies heavily on a complex array of secreted protein toxins [3]. Based on their mode of action, these toxins are classified into four major functional groups: membrane-damaging enzymes, pore-forming toxins (PFTs), intracellular toxins, and hydrolytic enzymes [41,42,43,44].

3.1. Alpha Toxin (CPA)

Alpha toxin (CPA), encoded by the chromosomal cpa/plc gene, is a phospholipase C with well-established membrane-damaging activity [28,43]. Its complex mode of action involves binding to the target plasma membrane via the TrkA receptor, triggering a signal transduction cascade that activates protein kinase C and stimulates the arachidonic acid cascade [43,45,46]. Hydrolysis of phosphatidylcholine and sphingomyelin produces diacylglycerol and releases inflammatory mediators (including prostacyclin, thromboxane, leukotrienes, and platelet-agglutinating factors), leading to acute tissue damage and cardiovascular disruption [45,46]. CPA also acts synergistically with sialidases (which remove sialic acid to expose gangliosides) and perfringolysin O to enhance membrane toxicity and promote vascular leukostasis [22,47,48,49,50].

Historically, CPA was considered a major determinant of avian NE. However, experimental disruption of plc in a virulent chicken isolate did not abolish disease-producing capacity, proving that CPA is not essential for NE development in chickens [51]. Consequently, its enzymatic and tissue-damaging activities should be distinguished from an indispensable causal role, and its contribution likely depends on synergistic interactions with other virulence determinants.

3.2. NetB Toxin

NetB is a member of the Staphylococcal α-hemolysin family of β-pore-forming toxins (β-PFTs) [52]. It forms heptameric, hydrophilic pores with an interior diameter of approximately 26 Å that strongly prefer cations [24,53]. It promotes osmotic cell lysis by allowing Na+, Cl⁻, and Ca²⁺ to enter the cytoplasm [54,55]. Expression of netB is strictly regulated by the VirR/VirS two-component system [56,57] and an accessory agr-like quorum-sensing system [1,58].

NetB is the primary virulence determinant in C. perfringens type G infections. Early functional studies established its essential role, showing that disruption of netB in a virulent isolate eliminated disease-producing capacity, whereas complementation restored virulence [59]. Epidemiological surveys consistently demonstrate an enrichment of netB-positive and NetB-producing strains among NE-affected flocks [60,61]. However, type A or netB-negative strains can occasionally cause NE [62,63,64], while healthy chickens may harbor type G strains [65]. Furthermore, in vivo evaluations using ligated-loop models identified both virulent netB-negative isolates and low-virulence netB-positive strains [66]. Because non-pathogenic chicken strains are phylogenetically distinct from pathogenic avian and mammalian isolates [67], netB carriage alone cannot reliably define virulence. Instead, NE pathogenesis reflects the combined influence of NetB expression, strain background, regulatory networks, and host intestinal predisposing factors [66,68]. Therefore, NetB is best described as a major experimentally supported virulence determinant rather than a standalone diagnostic marker [66,68].

3.3. Perfringolysin O (PFO)

Perfringolysin O (PFO), encoded by pfoA, is a cholesterol-dependent cytolysin (CDC) produced by nearly all C. perfringens strains [37,69]. PFO specifically targets eukaryotic cell membranes requiring high cholesterol concentrations [70], progressing through membrane binding, cholesterol recognition, oligomerization, and insertion to form large amphipathic pores [71]. At sublethal concentrations, PFO significantly upregulates intracellular adhesion molecule 1 (ICAM-1) and CD11b/CD18 glycoprotein in endothelial cells, promoting leukocyte adhesion, local edema, and ischemia [48,49]. At high concentrations, PFO exhibits leukocytotoxic effects, disrupting endothelial integrity and causing systemic shock and multiorgan failure [49,50]. Deletion of CDC genes significantly reduces bacterial pathogenicity, highlighting CDCs as potential therapeutic targets [72].

3.4. Beta2 Toxin (CPB2)

Beta2 toxin (CPB2) is a plasmid-encoded 28 kDa cytolytic pore-forming toxin sharing functional sequence homology with β-toxin (CPB) [5,73,74]. First identified in a piglet with enteritis [74], cpb2 has since been detected across multiple host species [75], encoded by either “consensus” or “atypical” gene variants [76,77]. In vitro, CPB2 induces cell rounding and lysis at concentrations >20 μg/mL via membrane disruption [78]. Although cpb2 carriage is documented in diseased animals [79], cpb2-positive strains are also frequently recovered from healthy livestock and poultry [76]. In chickens, available evidence supports a potential contributory role rather than an independently established causal role in NE. Thus, detection of cpb2 should be reported as a virulence-associated genotype unless active toxin expression and functional activity are demonstrated [32,80].

3.5. TpeL Toxin

TpeL is a 205 kDa plasmid-encoded toxin belonging to the large clostridial glucosylating toxin family [41,81], making it the largest toxin produced by C. perfringens [82]. Produced during vegetative growth or sporulation [81], TpeL expression is suppressed by glucose and sucrose [40] and is sensitive to trypsin digestion [81]. TpeL binds to the host cell-surface receptor LRP1 (low-density lipoprotein receptor-related protein 1) [83] and utilizes its transmembrane and catalytic domains to deliver a glycosyltransferase enzyme into the cytoplasm, inactivating Ras signaling and triggering apoptosis [84,85].

While tpeL carriage alone is insufficient to cause NE [86], experimental broiler models indicate that NetB-positive/TpeL-positive isolates exhibit greater disease severity, faster infection kinetics, and higher mortality rates [82,87,88,89]. Comparing NE-producing isolates to non-pathogenic strains shows a higher co-prevalence of cpb2, netB, and tpeL [90]. TpeL is therefore regarded as a potent virulence-enhancing factor whose synergistic interaction with NetB remains an active area of investigation [82,89].

4. C. perfringens Virulence Factors

Several virulence factors of pathogenic C. perfringens result in NE progress in chickens. These virulence factors play a critical role in toxin generation, adhesion of bacteria to the mucosa (adhesins), and provision of nutrition for their fast multiplication (degradative enzymes). Several genes and genetic variants, including netB, pfoA, cpb2, tpeL, and cna-associated loci, have been reported at higher frequencies or associated with NE-producing isolates; however, their detection alone does not establish expression or an independent causal role in disease [16,91]. Several C. perfringens proteins, such as collagen adhesion protein (CNA), have been suggested to act as adhesins [92]. C. perfringens adherence to extracellular matrix proteins plays a significant role in the pathogenesis of NE and is highly associated with their virulence [93]. The adhesin-encoding gene cnaA, located in the VR-10B chromosomal region, is essential for the pathogen to adhere to gelatine and collagen types IV and V [94]. Additionally, C. perfringens generates an adhesive pilus that is necessary for adhering to the intestinal wall. This pilus is made up of three components, CnaA, FimA, and FimB, which are encoded in the VR-10B chromosomal region [94,95]. C. perfringens fibrinogen-binding proteins FbpA and FbpB target the extracellular matrix protein fibronectin to promote host cell interaction and subsequent colonization [16,96] (Table 3).

Table 3.

C. perfringens virulence factors.

Virulence Factor	Gene	Location	Functional Role	Evidence in Poultry NE	Interpretation	References
NetB	netB	Plasmid/NELoc-1	Pore formation	Strong experimental support	Major virulence determinant	[59,60,66]
CPA	cpa/plc	Chromosome	Phospholipase/membrane injury	Present, but not essential in knockout model	Contributory	[45,46,47]
PFO	pfoA	Chromosome	Cholesterol-dependent pore formation	Virulence-associated	Contributory	[48,49,50,70,71,72]
CPB2	cpb2	Plasmid	Cytotoxic/pore-forming activity	Association inconsistent	Candidate	[73,74,75,76,77,78,79,80]
TpeL	tpeL	Plasmid	Glucosylating toxin	Associated with increased severity in some strains	Virulence enhancer/candidate	[82,86,87,88,89,90]
CnaA	cnaA	Chromosome	Collagen adhesion	Experimental adhesion/virulence evidence	Adhesin	[92,93,94,95]
FimA/FimB	Corresponding pilus locus	Chromosome	Type IV pilus-mediated adhesion	Experimental evidence	Colonization	[94,95]
FbpA/FbpB	Respective loci	Chromosome	ECM/fibronectin interaction	Association/functional evidence	Adhesion	[16,96]
NanI/NanJ/NanH	nanI/J/H	Chromosome	Mucin/sialic-acid utilization	Mechanistic evidence	Nutrient acquisition/colonization	[97,98,99,100,101,102]
ZmpA/ZmpB	zmpA/zmpB	Plasmid/chromosome	Mucin degradation	Virulence attenuation evidence	Colonization/tissue invasion	[102,103,104,105]

NE, necrotic enteritis; ECM, extracellular matrix.

Sialic acid (N-acetyl neuraminic acid) is a component of the mucus glycoproteins [97]. Certain infections, such as influenza viruses, Vibrio cholerae, Streptococcus pneumoniae, and C. perfringens, generate sialidase enzymes that, when they release sialic acid, may provide these microbes with nutrients [98,99]. In order to produce nutrients for its growth, C. perfringens develops a variety of sialidases, including NanH, NanI, and NanJ, which improve its adherence to intestinal tissues and break the sialic acid connections at various sites [100,101]. Furthermore, intestinal damage caused by bacterial toxins induces goblet cells to secrete more mucus, which could increase the growth of harmful organisms [102]. Zinc metalloproteases, or mucinases, are additional virulence factors that are expressed by the zmpA and zmpB genes. The zmpA gene is found within the NELoc-1 plasmid-encoded region, which also encodes NetB, but the zmpB gene is found on a chromosome [103,104]. Proteases are primarily secreted by bacteria that live on or in mucosal surfaces. They facilitate pathogen colonization by breaking down mucin, the main glycoprotein element of mucosa [102,105]. The theory states that both proteases participate in the same catabolic process that enhances virulence, and absence of one protease is enough to stop the process and reduce virulence [103] (Table 3).

Recent studies reinforce the view that NE virulence cannot be inferred from the presence of individual toxin genes alone. Contemporary phenotypic, genomic, and transcriptomic analyses indicate considerable strain-level heterogeneity in adhesion, toxin-associated pathways, metabolic adaptation, and expression of virulence-related genes during infection. In particular, in vivo classification of diverse poultry isolates has demonstrated discordance between netB carriage and lesion-producing capacity, while more recent infection-associated transcriptomic analyses indicate dynamic regulation of bacterial genes during host colonization. These findings support an integrated model in which virulence emerges from strain genetic background together with environmentally responsive gene expression rather than from a single toxinotype marker [66,106] (Table 3).

5. Pathogenesis of C. perfringens in Poultry

At the molecular level, pathogenesis describes the intricate and dynamic interactions between hosts and pathogens [58,107]. Understanding the pathogenesis is important for devising control measures scientifically [108]. The six stages of the pathogenesis of a fast-growing bacterial pathogen such as C. perfringens are: (1) colonization of the site of preference; (2) growth and proliferation; (3) nutrition acquisition; (4) evasion of host defense mechanisms; (5) damage to host tissues; and (6) transmission [54] (Figure 2).

Figure 2.

Multi-stage molecular pathogenesis of C. perfringens in poultry intestine. C. perfringens pathogenesis in poultry proceeds through six distinct stages: colonization via adhesion and bacteriocin-mediated commensal displacement; proliferation fueled by nutritional mucolysis (exacerbated by coccidiosis-induced mucin synthesis); and subsequent Agr-like quorum sensing and other regulatory networks contribute to modulation of virulence-associated gene expression. Upon establishment, the pathogen breaches the mucous layer, secreting pore-forming toxins and sloughing the epithelium. C. perfringens then targets the lamina propria with proteolytic enzymes, establishing a persistent Agr-regulated biofilm with alpha-toxin and perfringolysin, leading to deep tissue necrosis and subsequent environmental transmission. QS, quorum sensing.

Mucins are secreted by intestinal epithelial cells, which provide a physical barrier that prevents infections from entering [109]. To establish and promote the progression of NE, C. perfringens must colonize and degrade the mucus layer [110]. Potential locations for bacterial adhesin binding are provided by oligosaccharides on the mucin glycoproteins’ core glycosylated region [111]. Bacteriocins secreted by pathogenic C. perfringens, such as perforins encoded by the cpp gene, have the ability to displace commensal Clostridium species that are colonizing the intestinal mucosa [112,113]. C. perfringens has chitinases that can break down mucins and glycoside hydrolases that can use the oligosaccharide sugars as an energy source [1]. Mucus degradation in the small intestine is probably one of C. perfringens’ main sources of nutrition [114]. Predisposing factors such as coccidiosis induce a local T cell-mediated inflammatory response that promotes the synthesis of mucin and mucus, both of which are necessary for C. perfringens growth because of its mucolytic activity [115]. This enables the bacterium to generate the QS auto-inducing peptides that initiate the VirR/VirS cascade and create localized microcolonies on the mucosal surface [116] and virulence-related genes such as NELoc-1 involved in adhesion [58]. When the mucous layer breaks down, C. perfringens secretes pore-forming toxins that penetrate the epithelial cells. As C. perfringens spreads over the lamina propria and advances towards the intercellular junctions, it damages the basal domain of the epithelia with its proteolytic and collagenolytic enzymes. Thus, the intestinal epithelium sloughs off and becomes necrotic [117]. The development of C. perfringens biofilm on the exposed submucosa is facilitated by the Agr-like quorum sensing-regulated production of alpha toxin and perfringolysin [118].

Host responses are integral to NE pathogenesis and should be considered together with bacterial virulence mechanisms. Disruption of the mucus layer and epithelial junctional architecture compromises intestinal barrier integrity, increasing paracellular permeability and exposing underlying tissues to bacterial products [119]. Experimental NE models have demonstrated altered expression of genes associated with tight-junction organization, inflammatory signaling, apoptosis, and epithelial homeostasis, indicating that lesion formation reflects both direct toxin-mediated injury and active host cellular responses. Pore-forming toxins can disturb membrane integrity and ionic homeostasis and may trigger inflammatory cell-death pathways, while epithelial injury is accompanied by changes in junctional proteins, mucosal immune signaling, and tissue repair responses [120]. Recent experimental studies further demonstrate measurable increases in intestinal permeability following C. perfringens challenge, particularly when epithelial injury is potentiated by predisposing infections such as Eimeria. Several mechanistic insights have elucidated that pathogen-induced damage involves precise host epithelial remodeling, apoptotic cascades, and structural or functional dysregulation of membrane efflux complexes, particularly ATP-binding cassette (ABC) transporters [121,122,123]. Consequently, NE should be viewed as a dynamic host–pathogen process where bacterial colonization and toxin activity act together with host barrier failure, transport dysregulation, and regulated cell-death pathways [124].

Recent experimental evidence has expanded the understanding of host epithelial injury during C. perfringens infection. In broilers, C. perfringens challenge has been associated with impaired jejunal morphology, reduced expression of tight-junction components including occludin and claudin-1, and increased expression of apoptosis-associated genes such as Caspase-3, Caspase-9, and Bax [15]. Complementary mechanistic studies of PFO have shown disruption of epithelial junctional proteins, mitochondrial dysfunction, reactive oxygen species generation, and activation of pyroptotic responses. Collectively, these findings indicate that NE-associated tissue damage involves not only extracellular toxin-mediated membrane injury but also disruption of epithelial barrier homeostasis and regulated host-cell death pathways [120].

Contemporary NE challenge models have further demonstrated that disease phenotype reflects interactions among strain virulence, predisposing factors, intestinal microbial ecology, and host responses rather than bacterial abundance alone. Recent broiler studies comparing clinical and subclinical NE have linked disease severity with changes in jejunal microbial communities and intestinal health, reinforcing the multifactorial nature of NE pathogenesis [14,125].

6. Antimicrobial Resistance of C. perfringens Recovered from Poultry

Control strategies for NE involves the use of antimicrobials [126]. Antimicrobials have been used for many years to prevent NE and increase broiler production [127]. Antimicrobials are either used for therapeutic or preventative purposes in animal feed [128]. The use of antimicrobials in the poultry industry raised concerns regarding their continued usage in the antimicrobial growth promoters (AGPs) and their contribution to the development of antimicrobial resistance in bacteria [129]. Antimicrobial resistance is also a concern for C. perfringens infections. The prolonged and extensive use of antibiotics in poultry over the past few years has altered the bacterial environment, eliminated susceptible strains and promoted the persistence and dominance of antimicrobial-resistant bacteria [130]. For many years, antibiotics were used to promote growth; however, this practice has since been banned in a number of countries [131] (Figure 3).

Figure 3.

Emergence of multidrug-resistant (MDR) C. perfringens in poultry. Antimicrobial exposure, including therapeutic use, preventive applications, and historical antimicrobial growth promoter (AGP) usage, creates selection pressure that promotes the emergence and persistence of resistant C. perfringens strains. MDR isolates exhibit resistance through combined phenotypic profiles and genetic determinants, including tet/TetK, erm(B), bla, inu, and qnr, associated with resistance to tetracyclines, macrolides, beta-lactams, lincosamides, and fluoroquinolones, respectively. This highlights the increasing challenge of controlling C. perfringens-associated necrotic enteritis under antibiotic-restricted production systems.

Different investigations have reported multidrug resistance in C. perfringens isolates [132]. C. perfringens of foodborne illnesses has been found to be resistant to tetracycline, lincomycin, enrofloxacin, cefoxitin/ampicillin, and erythromycin through the existence of tet, inu, qnr, bla, and erm(B) genes, respectively, in Egypt [133]. Another study showed that absolute resistance to erythromycin, spectinomycin, and sulphamethoxazole + trimethoprim was demonstrated in 30 C. perfringens isolates (18 from chickens, 9 from rabbits, and 3 from calves). These isolates exhibited a high sensitivity of 90% to ampicillin + sulbactam, a moderate sensitivity of 73% and 63% to ofloxacin and cefotaxime, respectively, and 50% to each of meropenem and cefaclor. For amoxicillin + clavulanic and colistin, the isolates’ lowest sensitivity percentages were 43% and 40%, respectively. According to the Egyptian authors Eman and Sharaf [134], all examined C. perfringens type A isolates were multidrug resistant (MDR) showing resistance against all tested antimicrobial disks, including cefoxitin, tetracycline, gentamicin, amoxicillin + clavulanic acid, clindamycin, and sulphamethoxazole-trimethoprim (cotrimoxazole). Consequently, to validate these phenotypic findings, antimicrobial resistance genes to oxytetracycline and amoxicillin–clavulanic acid (Bla and TetK) were examined (Figure 3).

7. Biofilm Formation and Biofilm-Associated Antimicrobial Tolerance

Antimicrobial resistance should be distinguished from biofilm-associated tolerance and persistence. Antimicrobial resistance generally refers to a stable capacity to grow at antimicrobial concentrations that inhibit susceptible organisms and may result from acquired or intrinsic resistance determinants. In contrast, biofilm-associated tolerance reflects the enhanced survival of biofilm-embedded cells during antimicrobial exposure without necessarily involving a corresponding heritable increase in antimicrobial resistance. Persistence represents the survival of a small phenotypic subpopulation during otherwise lethal antimicrobial exposure. Restricted antimicrobial diffusion through the extracellular matrix, altered metabolic states, stress responses, and heterogeneous growth conditions may contribute to biofilm-associated tolerance but should not themselves be described as antimicrobial resistance.

In addition to its ability to produce toxins, C. perfringens is able to form biofilms, which increase bacterial resistance to disinfection and contributes to its continuing existence in the environment [135]. Biofilms can be described as the houses and cities of the bacterial world [136]. They are defined as “aggregates of microorganisms in which extracellular polymeric substances (EPSs) that are adhering to a surface and/or each other’s cells are frequently embedded in a self-produced matrix” [137] that undergo stages of accumulation, attachment, maturation, and dispersal [138]. Proteins, lipids, carbohydrates, and DNA make up the EPS that supports complex biofilm; each of these components increases resistance to antibiotics [139]. It was found that C. perfringens biofilm contained extracellular DNA, proteins, and polysaccharides for the matrix. Carbohydrates and proteins had already been identified as elements of C. perfringens biofilms [140,141]. A study of C. perfringens biofilm composition demonstrated β-1,4-linked polysaccharides and extracellular DNA as components of the biofilm matrix [140].

Biofilm-associated cells can display substantially reduced antimicrobial susceptibility compared with their planktonic counterparts [140,141], resulting in increased resistance to antimicrobial drugs and reduced effectiveness of biofilm-related infection treatment. However, the magnitude of this difference varies according to the bacterial species, antimicrobial agent, biofilm maturity, and experimental endpoint. Reports of very large fold differences originate largely from studies of other bacterial species and should not be generalized quantitatively to C. perfringens in the absence of directly comparable minimum inhibitory concentration (MIC), minimum biofilm inhibitory concentration (MBIC), or minimum biofilm eradication concentration (MBEC) measurements [142,143]. Bacteria can adhere to substrates and encapsulate themselves in a matrix consisting of extracellular DNA (eDNA), proteins, and EPS when they form biofilms [144,145]. Microorganisms’ surface is shielded from harm by EPS, which is the primary structural element of biofilms [146]. A linear glucosamine polymer called polysaccharide intercellular adhesin (PIA) makes up the majority of polysaccharide slime, which promotes bacterial cell attachment and biofilm formation [147]. When it comes to bacterial adhesion, extracellular DNA is essential for both the formation and maturation phases of biofilm development [148]. The combination of secreted extracellular proteins, protein subunits of cell appendages including pili and flagella, cell surface adhesions, and outer membrane vesicle proteins makes up the extracellular proteins found in the biofilm matrix. Their interactions with exopolysaccharides and nucleic acid constituents aid in the stability of the biofilm matrix, surface colonization, and preservation of the biofilm’s integrity and structure [143,149]. The biofilm layer gives the bacteria an ability to cause a variety of diseases. Thus, it is believed that bacteria that form biofilms can cause 65–80% of infections [150].

Several regulatory mechanisms play a role in regulating gene expression when C. perfringens forms biofilms. A total of 25.7% of the genes in C. perfringens, including those involved in EPS production (epsA-O, tasA, tapA, bslA, sipW, and calY), sporulation (sigG, sleC, soj, spoOA, spoVB, spoVD, and spoIIE), motility (ccpA), toxin production (virX, luxS, codY, and ctrAB), and the stress response (hrcA, lexA, and recA) show differential expression between biofilms and planktonic cells [151]. The downregulation of virulence genes encoding alpha-clostripain, collagenase, netB toxin, perfringolysin O, and phospholipase C indicates that the biofilm induces a quiescent growth phase and that the toxin proteins may not be directly involved in the creation of the biofilm [152]. It was proved that the type IV pilus and the catabolite regulatory protein CcpA are essential for C. perfringens to generate maximal biofilms [141,153]. However, hemolysins (such as perfringolysin O and alpha toxin) were found in a compound with eDNA and were increased in the biofilm, suggesting a possible increase in its pathogenicity [118]. The expression of toxin genes is probably downregulated during C. perfringens biofilm formation at optimal growth temperatures, but at lower temperatures, toxin genes might be involved in the synthesis of extracellular matrix to strengthen the bacterium’s adaptability to the altered environment [153] (Figure 4).

Figure 4.

Biofilm formation by C. perfringens isolates. C. perfringens biofilms develop through a structured five-stage process; attachment, adhesion, growth, maturation, and detachment forming complex “bacterial cities” encased in a protective matrix of eDNA, proteins, and polysaccharides. This self-produced extracellular polymeric substance (EPS) acts as a physical shield contributing to environmental persistence. Biofilm-mediated infections are further regulated by a quiescent growth phase and genetic shifts in sporulation and matrix production, resulting in high rates of multidrug resistance (MDR) and extensive drug resistance (XDR) in clinical and environmental isolates. EPS, extracellular polymeric substance.

In Egypt, genotyping of thirty C. perfringens isolates (20 diseased and 10 from chicken carcasses) revealed genotype A (cpa+). The virulence genes cpe, cpb2, netB, and tpeL were detected with percentages of 6.7%, 56.7%, 56.7%, and 36.7%, respectively. Eight isolates (26.7%) were XDR, one isolate (3.3%) was PDR, and twenty-one isolates (70%) were MDR. A multiple antibiotic resistance index of 0.7 was averaged. Of the C. perfringens isolates, 63.3% formed biofilms, which were divided into three categories: weak (36.7%), moderate (16.7%), and strong (10%). C. perfringens biofilms were significantly reduced by sodium hypochlorite [154]. In another Egyptian study, fifty C. perfringens isolates from various sources were tested for antibiotic susceptibility, and the results showed that 82–94% of the isolates were resistant to penicillin, cefotaxime, cefoxitin, ceftriaxone, clindamycin, and chloramphenicol. On the other hand, 86% of the isolates showed metronidazole susceptibility and all of them were susceptible to vancomycin. In addition, 74% of the studied isolates were ampicillin, amoxicillin, and ampicillin-sulbactam sensitive. Ninety-two percent of the isolates (46/50) showed multiple drug resistance, and the multiple antibiotic resistance (MAR) index had an average of 0.63 and ranged from 0.28 to 0.9. All C. perfringens isolates were examined for biofilm production; of these, 14 (28%) were considered strong biofilm producers, which were categorized as XDR with a MAR index of ≥0.7, 60% (30/50) were moderate biofilm producers, which considered MDR with MAR indices ranging from 0.2 to 0.6 [155].

More recent studies have extended C. perfringens-specific biofilm research beyond descriptive characterization toward targeted biofilm control. For example, contemporary poultry-associated studies have demonstrated isolate-dependent biofilm phenotypes and evaluated thymol as a direct inhibitor of C. perfringens biofilm formation. These findings support the need to evaluate candidate interventions using organism-specific biofilm models rather than extrapolating antibiofilm mechanisms from unrelated bacterial species [156].

As shown in Figure 4, the five steps in formation and development of biofilm are as follows: (1) reversible attachment, (2) irreversible adhesion, (3) growth, (4) biofilm maturation, and (5) detachment [157,158]. In the first stage, free-floating bacteria attached firmly to the substrate surface. The production of extracellular polymeric materials, proteins, and other biomolecules by bacteria during the colonization stage of biofilm development increases adhesion strength. Microorganisms in the biofilm use QS to interact with one another as they mature. The microorganisms separate from the biofilm and attach to the surrounding media during the last stage of biofilm formation [159]. The detachment is caused by various factors, and these factors are external disturbance, endogenous enzyme depredation, release of EPS, surface binding proteins and starvation [160,161,162]. Fimbriae, flagella, and capsules are produced by bacteria in biofilms to help them adhere firmly to surfaces [149].

8. Mechanisms Contributing to Biofilm-Associated Antimicrobial Tolerance

Available evidence indicates that C. perfringens biofilms contain extracellular DNA, proteins, and polysaccharide components, and that type IV pili and regulatory factors such as CcpA contribute to biofilm development. These mechanisms are supported by direct studies of C. perfringens [140,141,142,143,144,145,146,147,148,149,150,151,152,153]. In contrast, several mechanisms frequently described in other bacterial biofilms, including specific exopolysaccharides, matrix-associated antibiotic-inactivating enzymes, and particular efflux or cation-response systems, have not been adequately demonstrated in C. perfringens. Such mechanisms should therefore be considered general conceptual models rather than established components of C. perfringens biofilm physiology.

For several decades, the poultry industry has regularly utilized subtherapeutic dosages of antibiotics as AGPs in broiler chickens to improve feed efficiency (FE) and decrease disease incidence [163]. However, improper, and excessive use of antibiotics in livestock farming has resulted in the emergence of bacteria resistant to these drugs, which poses a serious risk to both human and animal health [164]. As a result, in animal agriculture, the use of AGPs was banned by the European Union in 2006 [165]. Consequently, there has been a growing interest in finding viable replacements for antibiotics as growth promoters in broiler chickens [166].

9. New Approaches Against C. perfringens Isolated from Poultry

The evidence supporting non-antibiotic interventions differs substantially according to the experimental endpoint. In this review, efficacy is therefore distinguished among: (i) inhibition or killing of planktonic C. perfringens; (ii) prevention of initial biofilm formation; (iii) disruption or eradication of established mature biofilms; (iv) reduction in NE severity in controlled poultry challenge models; and (v) effectiveness under commercial or field conditions. These endpoints should not be considered interchangeable, because in vitro antimicrobial or antibiofilm activity does not necessarily predict therapeutic efficacy or production benefits in vivo (Figure 5).

Figure 5.

Integrated non-antibiotic therapeutic strategies for the management of C. perfringens and necrotic enteritis in poultry. The management of C. perfringens in poultry utilizes a multi-pronged approach featuring probiotics, prebiotics, and organic acids that work collectively to restore gut microbiota, enhance intestinal morphology, and disrupt bacterial cellular physiology. Innovative biological and molecular interventions, such as bacteriophage cocktails and antimicrobial peptides, provide targeted bactericidal activity and non-selective membrane destabilization to neutralize virulent toxinotypes. Furthermore, green-synthesized nanoparticles and phytogenic compounds offer sustainable alternatives that inhibit biofilm formation and downregulate virulence genes, ultimately improving feed efficiency and reducing necrotic enteritis lesions. Ag NP, silver nanoparticles; AMPs, antimicrobial peptides; CNC-ZnO, cellulose nanocrystal–zinc oxide; CPQ, C. perfringens phage cocktail; DHFR/DHPS, dihydrofolate reductase/dihydropteroate synthase; MCFAs, medium-chain fatty acids; MDR, multidrug-resistant; NE, necrotic enteritis; NPs, nanoparticles; NTZ, nitazoxanide; PDR, pandrug-resistant; ROS, reactive oxygen species; ZnO NP, zinc oxide nanoparticle.

9.1. Probiotics

Live cultures of benign bacteria or yeast species, known as probiotics, are being used more frequently in poultry feeds in place of AGPs [167]. Probiotics from the genera Bacillus, Lactobacillus, Enterococcus, and others have been tested against C. perfringens both in vivo and in vitro [168]. Through competitive exclusion, they maintain a regular gut microbiota, which benefits health. Additional probiotics exhibit antagonistic properties towards infections, modify microbial metabolism, and generate advantageous metabolites, thus enhancing feed intake, digestion, and immune system performance [169,170]. By using a competitive exclusion mechanism, probiotics can also prevent harmful bacteria like Salmonella spp. and C. perfringens from growing and colonizing in the intestines [21]. Since probiotics have been shown to have a preventive effect on clostridial disease, it has been established that probiotic cultures possess significant antibacterial potential to be utilized as biological agents to prevent C. perfringens infections [171]. It has been demonstrated that adding probiotic Bacillus subtilis to broiler diets reduces the amounts of harmful bacteria in the GIT, enhances intestinal integrity and nutrient retention, and improves feed conversion [172,173,174]. A previous research on broilers with NE showed that B. subtilis increased the abundances of beneficial commensal microbes and associated compounds, which in turn improved growth performance and reduced intestinal inflammation [175,176]. Economic savings of $0.018 USD/kg of body weight were also possible with probiotics when B. subtilis was incorporated into a diet with a two percent reduction in metabolizable energy [177] (Figure 5).

While general probiotic mechanisms include restoration of the perturbed microbiota, production of antimicrobial molecules, competitive exclusion of pathogens from colonization, and host immune modulation, the precise mechanisms by which probiotics control C. perfringens-induced NE depend on a number of factors, including the species and strain of the probiotic agent, age and type of the bird, host immune status, and importantly, the severity of NE [21]. Probiotics and their mechanism of action to prevent NE in poultry are presented in Table 4.

Table 4.

Probiotics and necrotic enteritis prevention in poultry.

Genera	Bacteria (Probiotics)	Effect	Reference
Lactobacillus	Lactobacillus acidophilus	Controls the broiler microbiome and boosts beneficial microbes during C. perfringens exposure.	[178,179]
	L. acidophilus and L. fermentum	Reduce NE-induced inflammation, C. perfringens proliferation, and alpha-toxin production.	[180]
	L. acidophilus and L. casei	Decrease C. perfringens colonization, intestinal lesions, and NE-related mortality.	[181]
	L. fermentum 1.2029	Reduces NE lesion severity, IFN-γ levels, and toll-like receptor (TLR2) expression, while upregulating ileal IL-10.	[182]
	L. johnsonii	Suppresses C. perfringens-induced NE and exerts anti-inflammatory effects that protect broiler intestinal and liver tissues.	[183,184,185]
	L. plantarum and L. rhamnosus	Reduce pathogen-induced mortality along with intestinal Salmonella spp. and C. perfringens loads.	[186,187]
Clostridium	Clostridium butyricum	Stops NE progression in chickens by modulating immunity, barrier function, and gut microbiota, while improving weight gain and FCR.	[188,189]
Enterococcus	Enterococcus faecium	Optimizes gut microbiota composition, reduces inflammation, and enhances mucosal integrity in both healthy and C. perfringens-challenged broilers.	[190]
Bacillus	Bacillus subtilis	Improves bird performance, modulates gut microbiota and GALT activity, and prevents harmful bacterial colonization like Clostridium species.	[191,192,193,194]
	Bacillus licheniformis	Improves FCR, reinforces gut barrier integrity and GALT function, promotes balanced microbiota, produces bacteriocin-like compounds and lysozymes, and mitigates NE-associated oxidative stress.	[195,196,197]
	Bacillus amyloliquefaciens	Lowers pathogen load and lesion scores in E. tenella-, E. maxima- and C. perfringens-challenged broilers, while boosting Lactobacillus levels and FCR.	[198,199]
	Weizmannia coagulans (previously known as Bacillus coagulans)	Produces coagulin (a bacteriocin-like inhibitor) with potent activity against C. perfringens, inhibiting both its growth and alpha-toxin production.	[200,201,202,203]

α-toxin, alpha-toxin; IFNγ, interferon gamma; NE, necrotic enteritis; GALT, gut-associated lymphoid tissue; FCR, feed conversion ratio; E. tenella, Eimeria tenella.

9.2. Prebiotics

Prebiotics are more advantageous than probiotics because, in contrast to probiotics, they are designed to specifically support beneficial bacteria that are already present in the intestines [204].

Prebiotics include fiber, polyphenols, polyunsaturated fatty acids, and conjugated linoleic acid; they are health-promoting substrates for microorganisms [205,206]. Prebiotics are not able to be broken down and absorbed by fowl when given as a nutritional supplement. But they can also be very important for intestinal microecology, for encouraging the growth of specific bacteria, and for boosting nutrient absorption and utilization [207,208]. They also reduce the severity and duration of the lesions caused by C. perfringens in the intestine especially in jejunum and duodenum [209] (Figure 5).

A potential substitute for antibiotics has been investigated in Saccharomyces cerevisiae’s yeast cell wall (YCW) [210,211]. YCW extracts, which include mannan oligosaccharides (MOSs), mannan proteins, β (1,3)- and β (1,6)-glucans, chitin, and glycophospholipid surface proteins linked to the plasma membrane, have found numerous uses [212,213]. Therefore, feeding broiler chickens with dietary supplements comprising YCW extracts containing varying amounts of MOS and β-glucans may enhance gut morphology and growth performance, encourage the development of immune organs, increase the secretion of gut immunoglobulin, and hinder pathogenic bacteria from colonizing the gut [212]. It has been reported that dietary supplementation with MOS improves gut morphology, resulting in longer villi, shorter crypts, and more goblet cells [214]. MOS influenced immunological and metabolic pathways in the gut of infected chicks (Salmonella Typhimurium and C. perfringens) in a way that decreased colonization [215,216]. Dietary supplementation with β-glucans increased villi height in the gut and antibody levels against C. perfringens [217]. Supplementing chicks with β-glucans also enhanced the expression of genes related to innate and adaptive immunity, like β-defensins and cathelicidins, in their guts when they were challenged with NE [218,219,220].

9.3. Synbiotics

A probiotic and prebiotic combination is known as synbiotics; they work in concert to improve the colonization and survival of good bacteria in the digestive system [221,222]. Synbiotics help the host by producing antimicrobial chemicals, competitive exclusion, antibodies that modulate the immunological response, immune cells that enhance phagocytosis, natural killer cell activity, and anti-inflammatory cytokines [223]. In response to NE exposure, synbiotic supplementation enhances production performance, lowers lesion score, and stimulates a positive humoral and cell-mediated immune response [224]. The potential of a synbiotic feed additive including E. faecium, S. cerevisiae, and Bacillus species to prevent NE in broilers challenged with C. perfringens, a bacitracin-resistant pathogen, was assessed [225] (Figure 5).

9.4. Organic Acids

Organic acids (OAs) are carboxylic acids, which include fatty acids and have the chemical structure R-COOH along with certain other properties. Three classes can be distinguished from them: (a) simple mono-carboxylic acids, such as acetic, formic, propionic, and butyric acids; (b) carboxylic acids with hydroxyl groups, such as citric, tartaric, lactic, and malic acids; and (c) carboxylic acids with double bonds, such as sorbic and fumaric acids [226]. Specific lipid groups such as polyunsaturated, unsaturated, or saturated fats or fats classified as short-, medium-, or long-chain fatty acids based on the length of their fatty acid chains are the most often studied lipid groups in research. Short- and medium-chain fatty acids (SCFAs and MCFAs, respectively) have been shown to improve broiler health, production, feed digestibility, and reduce body and muscle fat [227,228] (Figure 6).

Figure 6.

Mechanistic basis of organic acids (OAs) as multi-target antimicrobial agents in poultry. Organic acids (OAs) improve the broiler health and intestinal integrity by promoting gastrointestinal cell proliferation, notably increasing villus height and crypt depth, while serving as potent non-antibiotic antimicrobial agents. Mechanistically, undissociated OAs penetrate the bacterial cell wall and dissociate intracellularly, causing a critical drop in internal pH that denatures proteins and exhausts cellular energy (ATP) via proton-pump activity. Simultaneously, the anionic fraction of the acid disrupts bacterial replication and translation by interacting with nucleic acids, effectively downregulating virulence factors and neutralizing pathogens like C. perfringens.

Because organic acids and their salts are usually regarded as safe, the European Union permitted their use in the production of chickens [229]. The benefits of organic acids are varied and include antibacterial properties, the promotion of intestinal integrity or gut development, enhanced nutritional digestibility, and an increased population of beneficial microbiomes (Lactobacillus spp.) [230]. Organic acids have antimicrobial properties as well as improving protein, energy, and mineral utilization in addition to increasing pancreatic output. Butyric acid exerted its highest effect on gastrointestinal cell proliferation, resulting in increases in villus height, surface area, and crypt depth [231]. Through increased intestinal barrier function, intestinal microbiota, and volatile short-chain fatty acid (SCFA) synthesis, dietary supplementation of various mixes of OA and/or phenolic compounds can mitigate the deleterious effects of NE challenge on intestinal health. When in-feed AGP is limited to use in chicken production, these results collectively show how OA mixes may be able to lessen the adverse effects of NE [232]. One possible mechanism of action for organic acids is non-dissociation, in which they penetrate the bacterial cell wall and modify the normal physiology of the bacteria, resulting in an internal pH change and the dissociation of H+ and anions, which consumes energy and may even kill the bacteria [8]. They also activate the intestinal mucosa, alter the microbiota, and have immunomodulatory effects. The type, dosage, and basis of the organic acid product determine the effects on broilers [233]. Lauric, capric-caprylic, and caproic acids are linked to reduced stool C. perfringens counts and enhanced gut histomorphology [234].

Antimicrobial Activity of Short-Chain Fatty Acids (SCFAs) or Medium-Chain Fatty Acids (MCFAs)

As mentioned above, in addition, the anionic component of the acid interacts directly with the free nucleic acids in the cytoplasm of the bacterium, impacting translation, transduction, and replication processes. This is because the bacterium lacks a nuclear membrane. Microorganisms’ pathogenic potential and the expression of resistance mechanisms would be negatively impacted by bacterial pathogenesis, which is dependent on the expression of virulence factors encoded at the DNA and which rely on transduction and translation mechanisms [235]. On the other hand, the acid’s cationic fraction (H+) lowers the bacterium’s internal pH, denaturing proteins and influencing enzyme activity. Because the pH affects the catabolic activity of the enzymes, a reduction in pH modifies the isoelectric point required for catabolic activation and lowers bacterial metabolism, resulting in bacteriostasis [236]. The bacterium uses membrane protein complexes (ATPases) to try and remove hydrogen ions to reverse the pH drop, but this requires a large energy expenditure, thus starting a bactericidal, biochemical, and negative physical cycle [235]. Since MCFAs have a greater pKa value than other organic acids, they are better at regulating microbial growth than they are at lowering pH [237,238]. Furthermore, a higher pKa value relative to the pH in the colon, small intestine, ventriculus, or gizzard will cause the balance to shift further towards the undissociated form, which will have a greater antibacterial impact [239]. The mechanism of action of organic acids and SCFAs as antimicrobials against C. perfringens is shown in Figure 6.

9.5. Bacteriophages

Bacteriophages infect and replicate inside a bacterial host and serve as natural biocontrol agents [240]. Bacteriophages and bacteria have a long-standing synbiotic and competitive relationship. In contrast to other antibiotic alternatives, bacteriophages are a unique class of living organisms that multiply by lysing the bacterium and automatically exploring the host’s surface-specific receptors [241,242].

Bacteriophages have four structural forms that make up their genetic material: ssDNA, dsDNA, ssRNA, and dsRNA. These forms are very adaptive to their surroundings. Bacteriophages select their genetic information; replication mechanisms based on several life cycle modes (lytic, lysogenic, and chronic) after they have entered the host bacterial strain [242,243]. Numerous studies have demonstrated the potential benefits of dietary bacteriophage supplementation in regulating intestinal microbial composition, improving mucosal immunity and the physical barrier function of the intestinal tract, and supporting intestinal microecological stability and normal animal body development [243]. Because of their comparatively high bactericidal activity, bacteriophages (phages) can damage bacterial host cells [244]. From chicken meat, phages targeting C. perfringens were successfully identified and given morphological and genetic characterizations. The significant potential of a 4-lytic phage cocktail (CPQ3, CPQ7, CPQ8, and CPQ10) as a natural biocontrol agent against C. perfringens in food and in vitro was documented [245].

As a potential substitute for the control and prevention of C. perfringens in chickens, a phage called HN02 has been demonstrated to be effective in reducing the pathogen’s contamination [246]. Thanki et al. [247] reported that out of the 32 recovered phages, 19 lysed 87–92% of the poultry strain collection worldwide (n = 97). In liquid culture, the pathogenicity of each of these individual phages, as well as 32 distinct phage combinations, was measured at various dosages. Next, in order to replicate an industry-used successful poultry model, a multi-strain infection model was created for C. perfringens larvae. Using the larva model, the effectiveness of 16/32 phage combinations was evaluated. This led us to conclude that, when given prior to bacterial challenge, the phage cocktail which included the phages CPLM2, CPLM15, and CPLS41 was the most successful at decreasing C. perfringens colonization in infected larvae. These findings imply that phages do provide a great deal of promise for treating and preventing C. perfringens infections in chickens. Mohd Shaufi et al. [248] reported that adding probiotics and phage cocktail to poultry diets has major favorable effects. In particular, the addition of 1 g/kg of probiotics and 1 g/kg of phage cocktail considerably enhanced the growth performance and decreased C. perfringens populations. A study in Egypt reported that NE in broilers could be effectively treated in experiments using the isolated podovirus C. perfringens phage [249].

9.6. Nanoparticles as Antimicrobial Agents Against C. perfringens

It has been demonstrated that nano-minerals or nanoparticles (NPs) improve growth, egg quality and production, immunological modulation, and antioxidant status for poultry. They also help to save production costs by lowering the need for additional mineral doses and increasing the feed conversion ratio. Additionally, they appear to be less hazardous than traditional mineral sources [250]. Consequently, nano-minerals may be used as mineral supplements as well as antimicrobial feed additives. In addition to nano-essential minerals, nanomaterials have the potential to modify the gut microbiome by directly preventing microbial development or by changing the way microbial metabolism occurs in the gut [251]. Eman and Sharaf [134] reported that nitazoxanide (NTZ) treats antibiotic-resistant C. perfringens-infected poultry without having a deleterious biochemical impact on liver or kidney functioning. Torky et al. [252] reported that Ag NPs have potent antibacterial action against MDR strains of C. perfringens. This study is the first, as far as the authors are aware, that examined the possible alternate antibacterial role of Ag NPs for the management of MDR C. perfringens in Egypt. Another study in Egypt demonstrated that 100 μg/mL of Ag NPs could prevent the production of biofilms in C. perfringens isolates recovered from humans, camels, and chickens. For the pigeon isolate, no biofilm formation was seen at a concentration of 75 μg/mL [155].

9.7. Antimicrobial Peptides as Antimicrobial Agents Against C. perfringens

Antimicrobial peptides (AMPs) are small protein molecules that are present in nearly all living things [253,254,255]. They have developed as a means of protecting the host against microbes and are crucial to innate immunity [256]. AMPs provide a number of benefits, including the capacity to function in multiple ways, ease of degradation in the environment, decreased accumulation, enhanced host immunity, neutralization of numerous microorganisms’ activities, and apparent low resistance frequency [254,257]. AMPs present broad-spectrum antimicrobial activity against bacteria, fungi, protozoa and viruses [258]. Because AMPs target non-selectively, they are more effective in eliminating MDR pathogens [258]. It is more difficult for bacteria to evolve resistance to the electrostatic interactions between AMPs and anionic cell membrane components than to develop resistance by modifying specific membrane targets. When it comes to their antibacterial, antifungal, and even antiviral characteristics, AMPs like lactoferrin are more adaptable (or non-specific) than antibiotics [259,260].

Electroporation, non-lytic membrane depolarization, membrane destabilization, pore formation, membrane thinning or thickening, and oxidized lipid targeting are some of the methods by which the majority of AMPs disturb the bacterial membrane [261]. However, certain AMPs can also interact with intracellular targets, interfering with bacterial metabolic turnover and limiting the synthesis of proteins, acids, and cell walls [262]. Because of their bactericidal or bacteriostatic properties, AMPs can be utilized to promote the broilers’ growth and to treat or prevent bacterial infections. In the avian gut, AMPs have been demonstrated to dramatically lower the number of pathogenic bacteria while simultaneously boosting the number of beneficial microbes and modifying the intestinal microbiota [254,263]. García-Vela et al. [264] reported that because of their potent activity against C. perfringens and other pathogens, synthetic enterocins, either alone or in combination, represent a promising alternative to antibiotics in the poultry industry. Pediococcus pentaceus FBB61 produces the bacteriocin pediocin A, which has shown antibacterial activity against C. perfringens type A [265]. In a different investigation, drinking water containing the peptide sublancin, which is generated by Bacillus subtilis 168, was given to chickens. The authors showed that this AMP might be employed as a possible antibacterial agent to manage C. perfringens-caused NE without altering the Lactobacillus community [266]. The food industry currently uses nisin, a bacteriocin generated by Lactococcus lactis, as an additive (GRAS, E234 in the EU) because of its ability to suppress potential bacterial infections [267]. Nevertheless, some studies have investigated its use in poultry. Numerous studies have shown that adding nisin to the diet alters the gut microbiota of the animal, lowering the potential of infections such as C. perfringens developing in the ileum [268]. García-Vela et al. [264] reported that a panel of twenty C. perfringens isolates was used to evaluate the inhibitory activity of a chemically synthesized set of enterocins from various subgroups of class II bacteriocins, tested individually and in combination, including class IIa enterocins A, P, and SEK4; the class IId leaderless enterocin L50; and the unsubgrouped class II enterocin B. The results showed that synthetic analogs of L50A and L50B were the most effective against C. perfringens.

9.8. Phytogenics as Antimicrobial Agents Against C. perfringens

Phytobiotic feed additives can be defined as plants or herbs that are added to animal feed to enhance animal performance. Herbs (non-woody, non-persistent plants), spices (boldly flavored), and essential oils (EOs) are examples of phytobiotics [269]. Phytogenic feed additives, or PFAs, have been shown in numerous studies to provide a range of benefits, including enhanced feed palatability, intestinal development, and health, as well as antibacterial, antiviral, antioxidant, antihelminthic, anti-inflammatory, and anti-stress responses [270]. Since the use of antibiotics in the feed industry is prohibited, they are mostly used as alternative AGPs in the production of nonruminant livestock (swine and poultry) [271]. Phytogenic compound (PC) can be utilized as extracts (crude or concentrated) or in solid, dried, and grinding forms [272]. Three primary groups of biologically active PCs have been identified: (1) phenolic compounds (flavonoids, phenolic acids, lignin, lignans, coumarins, stilbenes, and tannins); (2) terpenoids (plant volatiles, sterols, carotenoids, saponins, and glycosides); and (3) nitrogen-containing substances (alkaloids, glucosinolates, and cyanogenic glycosides) [273]. Considering the multitude of aromatic plants, herbs, and individual chemicals, their various modes of action are characterized by diversity and depend on their specific combination, concentration, and chemical composition.

The antibacterial activity of PCs is one of their remarkable qualities. Research on the antimicrobial qualities of PCs made from herbs and spices is quite comprehensive [274]. Thymol, carvacrol, eugenol, and cinnamon aldehyde are examples of plant-derived EOs that are frequently used as in-feed antibiotic alternatives to enhance the performance of livestock and poultry, boost intestinal health, and control chicken intestinal pathogens such as C. perfringens [275,276,277], because of their potent antioxidant, bactericidal, and antibacterial properties as well as their capacity to increase the secretion of intestinal digesting enzymes without leaving behind harmful residues [278]. When compared to antibiotics, EOs showed stronger antibacterial activity against C. perfringens in the in vitro investigation. When compared to untreated birds, the in vivo study revealed that birds treated with EOs had significantly lower bacterial counts, intestinal lesions, and improved performance [279]. Another study showed that supplementation with Scutellaria baicalensis and Lonicerae Flos (SL) extract could improve intestinal barrier function and histomorphology, hence increasing the growth performance of challenged birds and effectively mitigating the adverse impacts of C. perfringens challenge [280]. Furthermore, the research has shown that a combination of hydrophilic organic acids and hydrophobic EOs is more effective than either one alone at inhibiting harmful microbes [281,282]. It demonstrates beneficial effects on intestinal microbiota and immune regulation in chickens, strengthens intestinal barrier function, and eventually improves feed efficiency and growth performance in poultry [283], in addition to managing subclinical infections in chickens brought on by enteric pathogens such as Salmonella, E. coli, C. perfringens, C. jejuni, and Coccidia infections [284].

The potential synergistic effect of hydrophobic EOs and plant-derived organic acids could lie in their ability to impair the integrity of bacterial cell membranes, thereby facilitating the entry of hydrophilic organic acids into bacterial cells and their subsequent achievement of antibacterial and bactericidal effects [285,286].

9.9. Green Nanotechnology

Green synthesis, which uses the principles of green chemistry to create new nanomaterials, has the potential to drastically change industrial-scale nanomanufacturing processes [287]. Greener nanomaterials may play an increasingly important role in nanomedicine in the years to come as innovative drug delivery carriers and for other uses. Because green synthesis operates at room temperature and is easily scalable, it does not require costly equipment and is comparatively faster [288]. The process of creating nanoparticles with biomedical uses by utilizing microbes and plants is known as biosynthesis. This method is safe, green, economical, biocompatible, and environmentally friendly [289]. Green synthesis involves the use of bacteria, fungi, algae, plants, and other microorganisms. They enable the large-scale, impurity-free synthesis of ZnO NPs [290]. NPs synthesized using a biomimetic technique exhibit enhanced catalytic activity while reducing the need for costly and toxic chemicals [291]. The most favored source of NP synthesis is plants since they produce stable NPs with a wide range of sizes and shapes on large scales [292]. Wet chemically produced NPs have unique qualities, such as increased antibacterial efficacy of up to 99.9% when applied to cotton fabric [293]. Significant antibacterial activity has been shown by cellulose nanocrystal (CN)–ZnO bio-nanocomposites against a variety of Gram-positive and Gram-negative bacteria, such as Salmonella, Staphylococcus aureus, and Escherichia coli. The virulence toxin genes are efficiently downregulated by these composites, which lessens the bacteria’s capacity to cause diseases [294]. In addition to increasing the antibacterial effectiveness, the combination of CNs with ZnO NPs also improves ZnO’s stability and dispersibility, both of which are essential for real-world applications [295]. For the first time, adding CNs/ZnO composites to chicken feed or using them as a coating material may be a useful way to lower the incidence of bacterial diseases and enhance the general health and productivity of poultry [296].

The CNC/ZnO nanocomposites can physically interact with bacterial cell walls, resulting in destruction of the structure. This interaction is facilitated by the nanocomposites’ high surface area and sharp edges, which disrupt the cell membranes, leading to the leakage of intracellular contents [294,297]. When ZnO NPs are exposed to light, they are known to produce reactive oxygen species (ROS), which can harm the lipids, proteins, and DNA of bacterial cells. One important component of CNC/ZnO nanocomposites’ antibacterial action is oxidative stress [297,298]. According to molecular docking studies, CNC/ZnO nanocomposites have the ability to block important bacterial enzymes that are essential for bacterial survival and replication, including dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). This enzymatic inhibition further contributes to the antibacterial properties of the nanocomposites [298,299]. It has been demonstrated that the nanocomposites inhibit the expression of virulence genes in bacteria, hence decreasing the pathogenic potential of these organisms. These nanocomposites also have antibacterial properties through the control of gene expression [294].

By interfering with the extracellular polymeric materials that hold biofilms together, CNC/ZnO bio-nanocomposites have demonstrated effectiveness in inhibiting the formation of biofilms [294,300]. By penetrating biofilms and exhibiting antibacterial properties within the biofilm matrix, the bio-nanocomposites can decrease the viability of bacteria entrenched in biofilms [301]. Recent direct evidence has extended CNC/ZnO evaluation to PDR C. perfringens isolates recovered from poultry. Amin et al. [302] assessed both planktonic antimicrobial susceptibility and activity against established biofilms, thereby distinguishing antibacterial activity against free-living cells from effects on the biofilm phenotype. This represents a more relevant level of antibiofilm validation than studies demonstrating activity only against non-C. perfringens species; however, the evidence remains in vitro and requires confirmation in controlled poultry infection models before therapeutic or feed applications can be inferred.

Despite promising antimicrobial and antibiofilm activity, nanoparticle-based interventions require substantially more safety and translational evaluation before routine poultry application. Their biological behavior depends on composition, particle size, surface chemistry, dose, aggregation state, and route of administration. Potential concerns include cytotoxicity toward intestinal epithelial cells, accumulation or persistence of metallic components in edible tissues, disruption of beneficial gut microbial communities, and release of persistent nanomaterials into litter, soil, and water. Environmental exposure is particularly relevant for metal-based nanomaterials because repeated agricultural use may increase ecological metal loading and potentially contribute to co-selection pressures within environmental microbial communities. In addition, reproducible large-scale synthesis, batch-to-batch characterization, feed stability, cost-effectiveness, withdrawal requirements, residue limits, and regulatory approval remain unresolved for many experimental nano formulations. Accordingly, demonstration of in vitro antimicrobial or antibiofilm activity should be followed by pharmacokinetic, toxicological, residue-depletion, microbiome, environmental-fate, and controlled in vivo efficacy studies before field implementation.

The following Table 5 summarizes representative non-antibiotic interventions against C. perfringens in poultry, detailing their representative agents, experimental models, tested doses, primary outcomes, proposed mechanisms, and current limitations.

Table 5.

Comparative evidence for non-antibiotic interventions against C. perfringens and necrotic enteritis in poultry.

Intervention	Representative Agent	Experimental Model	Dose	Main Outcome	Proposed Mechanism	Evidence Level	Main Limitation	References
Probiotic	Bacillus subtilis PB6	CP-challenged broilers	107 CFU/kg feed	Improved growth and intestinal barrier; reduced challenge-associated intestinal injury	Competitive exclusion, microbiota modulation, barrier support	In vivo	Strain- and challenge-model dependent	[172,173,174]
Prebiotic	Yeast β-glucan	NE-challenged broilers	200 mg/kg diet	Improved intestinal health, immune response and CP control	Immunomodulation, barrier support, microbiota modulation	In vivo	Product composition and host response vary	[217,218,219,220]
Organic acids	OA/EO formulation	NE-challenged broilers	Formulation-specific; report exact inclusion used in primary study	Improved intestinal integrity and reduced NE-associated effects	Intracellular acidification, membrane effects, microbiota modulation	In vivo	Efficacy depends strongly on formulation, coating and dose	[232]
Phytogenic	Thyme EO	CP-challenged broilers	0.25% diet	Reduced intestinal CP counts and intestinal lesions	Membrane disruption and antimicrobial activity	In vitro + in vivo	Composition varies among plant sources/batches	[275,276,277,278,279]
AMP	Sublancin	CP in vitro and challenged broilers	MIC: 8 μM in vitro; 2.88, 5.76, or 11.52 mg /L of drinking water in vivo, administered fresh daily for 7 days	Direct inhibition and amelioration of experimental NE	Membrane/cell-envelope damage	In vitro + in vivo	Stability, production cost, delivery and resistance require evaluation	[266]
Bacteriophage	CP-specific phage cocktail	Experimental broilers	Study-specific phage titre	Reduced experimental NE severity/mortality	Host-specific bacterial lysis	In vivo	Narrow host range, phage resistance and delivery stability	[245,246,247,248,249]
Ag NPs	Silver nanoparticles	MDR/XDR CP isolates from poultry and other animal/human sources	25–100 μg/mL for antibiofilm; 100 μg/mL for biofilm inhibition. MIC: 20 μg/mL; MBC: 30 μg/mL	Inhibition of biofilm formation	Membrane damage/oxidative stress	In vitro	Toxicity/residue/environmental uncertainty	[155,252]
CNC/ZnO	Cellulose nanocrystal/ZnO nanocomposite	PDR, biofilm-producing CP isolates recovered from chickens and turkeys	MIC: 0.125 μg/mL; MBC: 0.25 μg/mL	Planktonic inhibition and reduction of pre-existing biofilms	Cell-envelope injury; agrB/pilA2 downregulation	In vitro	No equivalent therapeutic poultry/field validation	[302]

OA, organic acid; EO, essential oil; CP, C. perfringens; NE, necrotic enteritis; MDR, multidrug-resistant; XDR, extensive drug-resistant; PDR, pandrug-resistant; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration.

10. Future Perspectives and Research Gaps

Despite the considerable progress in understanding the pathogenesis and control of C. perfringens in poultry, several critical knowledge gaps remain. One of the major limitations is the incomplete understanding of the complex interplay between virulence factors, particularly toxin production [303], biofilm formation [151], and antimicrobial resistance [304]. While the netB gene has been widely recognized as a key virulence determinant, its presence alone does not fully explain disease occurrence, indicating that additional factors and regulatory mechanisms are involved.

Furthermore, the role of biofilm formation in C. perfringens pathogenicity is not yet fully elucidated. Although biofilms are known to enhance environmental persistence and resistance to antimicrobial agents, their direct contribution to disease severity and transmission under in vivo conditions requires further investigations. Standardized and reproducible methods for biofilm quantifications are also lacking, making it difficult to compare results across studies [156].

Another important gap lies in the variability of reported efficacy of alternative control strategies. Probiotics [21], prebiotics [305], organic acids [306], NPs, and AMPs [307] have shown promising results. However, their effectiveness is often strain-dependent and influenced by host factors, environmental conditions, and experimental design. There is a clear need for well-designed in vivo studies and large-scale trials to validate their approaches under field conditions [308].

In addition, limited information is available regarding the long-term safety, potential toxicity, and regulatory aspects of emerging interventions such as NPs and AMPs. The impact of these alternatives on the gut microbiome and their potential to induce resistance should also be carefully evaluated.

Future research should focus on integrative approaches combining genomics, transcriptomics, proteomics, and metabolomics to better understand the molecular mechanisms underlying C. perfringens virulence and adaptation [309]. Moreover, the development of target and synergistic strategies that simultaneously address toxin production, biofilm formation, and antimicrobial resistance may offer more effective and sustainable control of NE in poultry.

Overall, bridging these knowledge gaps will be essential for the development of innovative, safe, and effective alternatives to antibiotics in poultry production.

In conclusion, C. perfringens-associated NE remains a multifactorial challenge in poultry production in which toxin-mediated virulence, host susceptibility, intestinal dysbiosis, antimicrobial resistance, and biofilm-associated persistence can interact. Among non-antibiotic interventions, probiotics, particularly the selected Bacillus and Lactobacillaceae strains, together with several organic-acid, synbiotic, and phytogenic formulations, currently have the most substantial support from controlled poultry challenge studies. Bacteriophages and selected antimicrobial peptides have also demonstrated efficacy in experimental models, although delivery, spectrum, stability, and reproducibility remain important translational constraints. In contrast, nanoparticle and green-nanocomposite approaches show promising planktonic and antibiofilm activity but remain predominantly supported by in vitro evidence, with insufficient information on in vivo efficacy, residues, long-term safety, microbiome effects, environmental fate, and regulatory acceptability. Evidence for eradication of mature C. perfringens biofilms and large-scale commercial field efficacy remains limited across most intervention classes. Future studies should therefore prioritize standardized challenge and biofilm models, direct comparisons between planktonic and established-biofilm endpoints, well-controlled animal trials, and field validation incorporating safety, production performance, cost-effectiveness, and antimicrobial-resistance outcomes.

Author Contributions

Conceptualization, A.A. and N.K.A.E.-A.; data collection, I.A. and N.K.A.E.-A.; validation, I.A.; A.A.; I.P.; M.A. and N.K.A.E.-A., investigation, I.A.; A.A.; I.P.; M.A. and N.K.A.E.-A. data curation, I.A. and N.K.A.E.-A.; writing—original draft preparation, I.A. and N.K.A.E.-A.; writing—review and editing, I.A.; A.A.; and N.K.A.E.-A. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.