Antimicrobial Compounds from Anaerobic Microorganisms: A Review of an Untapped Reservoir

Abstract

Anaerobes, the oldest evolutionary life forms, have been unexplored for their potential to produce secondary metabolites due to the difficulties observed in their cultivation. Antimicrobials derived from anaerobic bacteria are an emerging and valuable source of novel therapeutic agents. The urgent need for new antimicrobial agents due to rising antibiotic resistance has prompted an investigation into anaerobic bacteria. The conventional method of antimicrobial discovery is based on cultivation and extraction methods. Antibacterial and antifungal substances are produced by anaerobic bacteria, but reports are limited due to oxygen-deficient growth requirements. The genome mining approach revealed the presence of biosynthetic gene clusters involved in various antimicrobial compound synthesis. Thus, the current review is focused on antimicrobials derived from anaerobes to unravel the potential of anaerobic bacteria as an emerging valuable source of therapeutic agents. These substances frequently consist of peptides, lipopeptides, and other secondary metabolites. Many of these antimicrobials have distinct modes of action that may be able to go around established resistance pathways. To this effect, we discuss diverse antimicrobial compounds produced by anaerobic bacteria, their biosynthesis, heterologous production, and activity. The findings suggest that anaerobic bacteria harbor significant biosynthetic potential, warranting further exploration through recombinant production for developing new antibiotics.

1. Introduction

The spread of infectious diseases from pathogenic microbes poses significant health risks, leading to increased antibiotic use. However, this overuse drives antimicrobial resistance (AMR), a serious global health threat linked to higher mortality in nosocomial infections [1,2]. Alarmingly, resistance is no longer limited to conventional antibiotics but has also emerged against alternative agents such as the FDA-approved antimicrobial peptide nisin, once considered a low-risk preservative [3]. These developments underscore the urgent and growing need to identify and develop novel antimicrobial agents capable of combating multidrug-resistant pathogens. Antimicrobial peptides (AMPs) offer a promising alternative to traditional antibiotics due to their broad-spectrum activity against bacteria, fungi, and viruses, and their ability to disrupt microbial membranes with reduced likelihood of resistance development [4,5].

Traditionally, antibiotic discovery has focused on aerobic soil bacteria, especially Actinomycetes and Bacillus species, which have yielded most clinically used antibiotics [1]. Researchers are expanding the search to unconventional microbial sources, including anaerobic bacteria. Anaerobic bacteria are well-established in industrial biotechnology for their roles in fermentation, production of solvents, and biofuels. Yet, their biosynthetic potential for antimicrobial production has long been overlooked, in part due to challenges in cultivation, genetic manipulation, and metabolite detection under oxygen-free conditions. While certain lactic acid bacteria (LAB) and Clostridium species have demonstrated the ability to produce bacteriocins and other bioactive metabolites [6,7], these represent only a fraction of the true biosynthetic potential harbored by anaerobes. Many of these organisms contain diverse and cryptic biosynthetic gene clusters (BGCs), previously dismissed as silent or inactive, but now revealed through genome mining and metagenomics to encode structurally novel antimicrobial compounds [6].

Recent advances in multi-omics, synthetic biology, and heterologous expression systems have reignited interest in anaerobes as promising reservoirs of bioactive natural products. Notably, genome-guided discovery has facilitated the identification of previously unknown BGCs in strict anaerobes, while metabolomics and transcriptomics are beginning to elucidate the regulatory networks controlling their expression. Metabolic profiling and genome mining have revealed that anaerobic bacteria harbor a wealth of BGCs associated with antimicrobial compound production [8]. Genome sequencing of Clostridium and other anaerobes has uncovered BGCs encoding ribosomally synthesized and post-translationally modified peptides (RiPPs), non-ribosomal peptides (NRPs), polyketides (PKs), and hybrid PKS–NRPS clusters [9,10]. For instance, the identification of a hybrid PKS–NRPS BGC in an anaerobic Epsilon-proteobacterium marked a novel finding, highlighting the untapped biosynthetic novelty within anaerobic lineages [11].

However, progress is hindered by significant research gaps, including the difficulty of cultivating fastidious anaerobes, limited compatibility of standard expression systems for anaerobic BGCs, and a lack of high-throughput screening methods tailored to anaerobic biosynthetic pathways.

This review aims to spotlight anaerobic bacteria as an underutilized but potentially rich source of antimicrobial agents. We explore the chemical diversity of antimicrobials produced by these organisms, the structure and regulation of their biosynthetic gene clusters, current strategies for cultivation and heterologous expression, and the reported bioactivities of anaerobe-derived compounds against clinically relevant pathogens. We also identify critical bottlenecks in discovery and development pipelines. By bringing these aspects together, we aim to underscore the untapped potential of anaerobes in antimicrobial drug discovery and propose strategic directions for future research.

We conducted a non-systematic literature review using databases such as PubMed, Scopus, and Google Scholar. No specific time restriction was applied; studies were included regardless of publication date, provided they offered relevant insights into antimicrobial compounds produced by anaerobic bacteria. Keywords used during the search included “anaerobic bacteria,” “antibiotics”, “bacteriocins,” “secondary metabolites,” “biosynthetic gene clusters (BGCs),” and “antimicrobial peptides.” Only peer-reviewed publications in English were considered. Priority was given to studies that included experimental validation (e.g., antimicrobial activity assays), genomic or bioinformatic characterization of BGCs.

2. Tools and Technologies for Unlocking Antimicrobial Potential in Anaerobes

2.1. Cultivation Strategies for Anaerobic Bacteria

Traditionally, the discovery of antimicrobial compounds from anaerobic bacteria relied on cultivating isolates under strictly anoxic conditions using specialized growth media supplemented with reducing agents and incubated within anaerobic chambers (Figure 1A). To maintain anaerobiosis, a range of physical and chemical methods are employed. Anaerobic jars use chemical sachets that generate hydrogen and carbon dioxide; the hydrogen reacts with residual oxygen in the presence of palladium catalysts to produce water, while redox indicators such as resazurin or methylene blue confirm oxygen depletion. For stricter control, anaerobic chambers (glove boxes) provide a fully enclosed, oxygen-free workspace maintained by a continuous flow of N₂/H₂/CO₂ gas mixtures, with internal palladium catalysts ensuring the removal of trace oxygen [12]. Culture media are pre-reduced with agents such as cysteine, sodium thioglycollate, or dithiothreitol (DTT) to maintain a low redox potential. Anaerobes are often grown in sealed, gas-tight tubes (e.g., Hungate or Balch tubes) flushed with anaerobic gas and sealed with butyl rubber stoppers [13]. More advanced techniques, including roll tubes, microfluidic culture platforms, and co-culture with hydrogen-scavenging organisms, are increasingly used to cultivate fastidious or unculturable strains. Together, these methods support the reliable isolation and growth of both facultative and obligate anaerobes for antimicrobial discovery.

These cultivation-based approaches enabled the isolation of a few antimicrobial-producing strains, particularly among LAB and Clostridium species. Once sufficient biomass is achieved, culture supernatants or cell extracts are collected to isolate active compounds. Techniques such as solvent extraction, solid-phase extraction, HPLC purification, and mass spectrometry are commonly used to identify and characterize antimicrobial metabolites [4,8]. Activity is typically confirmed using agar diffusion, broth microdilution, or co-culture inhibition assays. However, many anaerobes remain difficult to culture [14], and a large proportion of their secondary metabolite biosynthetic potential has likely gone undetected due to transcriptional silencing under native or laboratory conditions [15].

2.2. Genome Mining and Bioinformatic Tools

Recent advances in genomics and bioinformatics have drastically shifted the perspective of natural product discovery. To systematically explore this hidden diversity, researchers now employ tools such as antiSMASH [16] and BAGEL4 [17] to annotate and predict the function of BGCs from anaerobic genomes. These tools can identify complete BGCs and predict the types of secondary metabolites they are likely to produce. Moreover, curated repositories such as MIBiG [18] allow for comparison of newly identified BGCs against well-characterized reference clusters, facilitating dereplication and prioritization. Predictive tools such as PRISM [19] take this further by modeling the chemical structures of metabolites based on the encoded enzymes, especially within NRPS and PKS pathways. For peptides, dedicated databases like CAMP [20], APD [21], and SATPdb [22] catalog experimentally validated AMPs, while NPASS [23] and NPAtlas [24] provide additional information on compound structures, biological activities, and organismal sources. To contextualize the diversity revealed through genome mining, Table 1 summarizes biosynthetic classes of antimicrobial compounds discovered in anaerobes.

Despite their utility, these platforms were primarily developed for aerobic organisms and do not account for the unique metabolic constraints, redox sensitivities, or regulatory environments of anaerobic microbes. Currently, no dedicated databases or computational tools exist specifically for anaerobic bacteria, representing a key limitation in fully harnessing their antimicrobial potential.

2.3. Heterologous Expression of Anaerobic BGCs

To functionally access these cryptic BGCs, heterologous expression systems are increasingly employed (Figure 1B). Genes of interest are typically cloned and expressed in genetically tractable hosts such as Escherichia coli, Streptomyces, or engineered Bacillus strains. A significant challenge in characterizing microbial clusters is the lack of suitable heterologous hosts for expression. Common systems like Streptomyces and Bacillus subtilis are aerobes, and Escherichia coli performs poorly under anaerobic conditions, limiting the expression of anaerobic BGCs. To address this, a natural competence-based large DNA fragment cloning (NabLC) system has been developed for integrating over 70 kb genomic fragments into Streptococcus mutans UA159, a facultative anaerobic bacterium ideal for lab use [25]. This strain, part of the human oral microbiota, grows well anaerobically and has a fully annotated genome, making it capable of synthesizing various natural products. The NabLC platform successfully expressed several BGCs from anaerobic bacteria, including a pyrazinone cluster from Staphylococcus epidermidis and two novel BGCs from human oral Streptococcus. One cluster led to the discovery of the (2E)-decenoyl dipeptide (SNC1–465), while another produced mutanocyclin, an anti-infiltration tetramic acid. These findings position S. mutans UA159 as a promising host for exploring BGCs from anaerobic microbes, contrasting earlier focuses on aerobic genera like Actinomycetes and Myxobacteria [26].

3. Antibiotics from Anaerobes

Anaerobic bacteria are a valuable source of antibiotics with unique chemical structures and mechanisms of action. Bacterial strains pertaining to genera including Clostridium, Actinomyces (facultative anaerobic Streptomyces) produce antibiotics that have been employed to combat a wide range of pathogens, including drug-resistant strains [1,18]. However, the translational potential of these compounds is hampered by challenges in production scalability, synthetic accessibility, and low yields in native hosts [27]. For instance, clostrubin demonstrates potent anti-VRE activity but suffers from limited expression and complex biosynthesis. Additionally, in vivo studies remain scarce, and most compounds lack comprehensive pharmacokinetic or toxicity profiles. To harness their therapeutic potential, further work is needed in pathway engineering, fermentation optimization, and preclinical validation. Biosynthesis machinery of these antibiotics includes core biosynthetic genes, additional biosynthetic genes, and transportation related genes (Figure 2).

Table 1. Genomic properties, classifications, and accession information for selected antimicrobial biosynthetic gene clusters (BGCs) derived from anaerobic bacterial strains, including key compounds, producer strains, and biosynthetic class.

S.No.	Compound	Producer Strain	BGC Features (Genes/Size/Type)	Genomic Organization Summary	Classification	MIBiG Accession	Reference
1	Closthioamide	R. cellulolyticum DSM 5812	Multiple genes including ctaA–ctaK, noncanonical thiotemplated assembly line (~20 kb)	Modular genes for starter unit biosynthesis, sulfur incorporation, dimerization, and thioamidation; Rc-Sfp PPTase for PCP activation	Nonribosomal peptide-like thiotemplated metabolite	BGC0001891	[28]
2	Clostrubin	C. beijerinckii HKI0724	Type II PKS; ~40 kb aromatic polyketide cluster	Multi-module type-II PKS genes arranged with cyclases/oxidases for pentacyclic core formation	Polyketide	NA	[29]
3	Diffocins	C. difficile CD4, CD16	Phage tail-like BGC (~20 kb); ORFs CD1359–1376 including structural and receptor-binding proteins	Encodes R-type bacteriocin machinery; regulated by SOS response; ORF1374 determines target specificity	Phage tail-like bacteriocin (R-type)	NA	[30]
4	Circularin A	C. beijerinckii ATCC 25752	cirABCDE core genes; includes membrane (cirB, cirC), ATPase (cirD), and immunity (cirE)	Compact gene cluster with overlapping ORFs; circular bacteriocin via head-to-tail peptide bond	Head-to-tail cyclic peptide (Class V bacteriocin)	BGC0000488	[31]
5	Barnesin A	S. barnesii SES-3	Hybrid NRPS–PKS cluster (~20–30 kb); includes tailoring and regulatory enzymes	Modular gene layout with NRPS and PKS modules in tandem; expression validated by transcriptomics	Hybrid NRPS–PKS lipopeptide	BGC0001524	[11]
6	Ruminococcin C	R. gnavus E1	RiPP/sactipeptide-type BGC (~12.8 kb); includes 13 genes organized in 3 operons	Includes three structural genes for RumA, modification enzyme (RumM), and transporter (RumT); expression regulated by trypsin-dependent signaling	Type AII sactipeptide	BGC0002043	[32]
7	Estericin A	C. estertheticum CF016	RiPP cluster (~25 genes); includes radical SAM enzyme, protease, transporter	Lantibiotic-like BBGC7 cluster on ~180 kb plasmid; high stability against heat and enzymes	Class II lanthipeptide	BGC0002667	[33]

Table 1. Genomic properties, classifications, and accession information for selected antimicrobial biosynthetic gene clusters (BGCs) derived from anaerobic bacterial strains, including key compounds, producer strains, and biosynthetic class.

S.No.	Compound	Producer Strain	BGC Features (Genes/Size/Type)	Genomic Organization Summary	Classification	MIBiG Accession	Reference
1	Closthioamide	R. cellulolyticum DSM 5812	Multiple genes including ctaA–ctaK, noncanonical thiotemplated assembly line (~20 kb)	Modular genes for starter unit biosynthesis, sulfur incorporation, dimerization, and thioamidation; Rc-Sfp PPTase for PCP activation	Nonribosomal peptide-like thiotemplated metabolite	BGC0001891	[28]
2	Clostrubin	C. beijerinckii HKI0724	Type II PKS; ~40 kb aromatic polyketide cluster	Multi-module type-II PKS genes arranged with cyclases/oxidases for pentacyclic core formation	Polyketide	NA	[29]
3	Diffocins	C. difficile CD4, CD16	Phage tail-like BGC (~20 kb); ORFs CD1359–1376 including structural and receptor-binding proteins	Encodes R-type bacteriocin machinery; regulated by SOS response; ORF1374 determines target specificity	Phage tail-like bacteriocin (R-type)	NA	[30]
4	Circularin A	C. beijerinckii ATCC 25752	cirABCDE core genes; includes membrane (cirB, cirC), ATPase (cirD), and immunity (cirE)	Compact gene cluster with overlapping ORFs; circular bacteriocin via head-to-tail peptide bond	Head-to-tail cyclic peptide (Class V bacteriocin)	BGC0000488	[31]
5	Barnesin A	S. barnesii SES-3	Hybrid NRPS–PKS cluster (~20–30 kb); includes tailoring and regulatory enzymes	Modular gene layout with NRPS and PKS modules in tandem; expression validated by transcriptomics	Hybrid NRPS–PKS lipopeptide	BGC0001524	[11]
6	Ruminococcin C	R. gnavus E1	RiPP/sactipeptide-type BGC (~12.8 kb); includes 13 genes organized in 3 operons	Includes three structural genes for RumA, modification enzyme (RumM), and transporter (RumT); expression regulated by trypsin-dependent signaling	Type AII sactipeptide	BGC0002043	[32]
7	Estericin A	C. estertheticum CF016	RiPP cluster (~25 genes); includes radical SAM enzyme, protease, transporter	Lantibiotic-like BBGC7 cluster on ~180 kb plasmid; high stability against heat and enzymes	Class II lanthipeptide	BGC0002667	[33]

3.1. Closthioamide

Closthioamide, the first antibiotic isolated from an anaerobic bacterium, is non-ribosomally synthesized by Ruminiclostridium cellulolyticum (formerly Clostridium cellulolyticum). It exhibits potent activity against drug-resistant pathogens, including Enterococcus faecalis, Neisseria gonorrhoeae, and Staphylococcus aureus, with low MICs against methicillin-resistant Staphylococcus aureus (MRSA) and VRE, outperforming ciprofloxacin against the latter. It also shows moderate cytotoxicity [34]. Closthioamide is assembled via a unique thiotemplated biosynthetic pathway involving dimerization and thionation, distinct from canonical NRPS systems [28]. The ~20 kb gene cluster (ctaA–ctaK) encodes enzymes for starter unit biosynthesis, sulfur incorporation (thioamide formation), diaminopropane linker synthesis, and dimerization. Post-translational activation of carrier proteins relies on the Rc-sfp phosphopantetheinyl transferase located outside the core BGC. This cluster is registered in the MIBiG database as BGC0001891 [35]. Closthioamide inhibits bacterial DNA replication by targeting DNA gyrase and topoisomerase IV, specifically interfering with their ATPase activity while sparing the cleavage-rejoining function. It also blocks ATP-independent gyrase relaxation activity and shows no cross-resistance to ciprofloxacin or novobiocin in tested strains [36]. Structurally, closthioamide contains multiple thioamide moieties and functions as a copper chelator.

3.2. Clostrubin

Clostrubin is a purple-pigmented, pentacyclic aromatic polyketide discovered from Clostridium beijerinckii cultures. It features a unique benzo[a]tetraphene ring system and is the first polyketide identified from an obligate anaerobe, later confirmed by chemical synthesis [29,37]. Clostrubin is synthesized via a type II PKS system encoded in a ~40 kb biosynthetic gene cluster (BGC), which includes core PKS genes, cyclases, and oxidases responsible for assembling and modifying the aromatic scaffold. Although this BGC is not yet registered in MIBiG, its structure and function have been partially characterized [29]. Clostrubin exhibits potent activity against Gram-positive pathogens, including Mycobacterium spp., MRSA, and VRE, with MICs ranging from 0.12 to 0.97 μM, making it a strong candidate for further antimicrobial development [29].

3.3. Clostrindolin

Among small molecules, clostrindolin, a pyrone alkaloid antimicrobial substance, was isolated from the anaerobic bacterium C. beijerinckii [38]. It is a novel pyrone containing natural product with antimicrobial activity against M. vaccae. Therefore, this antimicrobial substance provides a natural platform to develop improved chemical moieties against pathogenic strains.

3.4. Naphthalecin

Naphthalecin is a novel polyketide antibiotic produced by the anaerobic soil bacterium Sporotalea colonica. It shows strong antibacterial activity against gram-positive bacteria and has cytotoxic effects at higher concentrations [39]. The compound was extracted and purified from bacterial cells using ethyl acetate and identified through reverse phase HPLC, LC-MS, NMR, and HRMS. Its name is derived from its unique naphthalene moiety.

4. Bacteriocins or AMPs from Anaerobes

Bacteriocins or AMPs are small, bioactive peptides produced by various aerobic and microaerophilic bacteria, essential for microbial competition and defense [40]. These peptides undergo posttranslational modifications for stability and possess unique mechanisms, like membrane disruption or inhibition of cell wall synthesis, making them promising for pathogen control [41]. However, these compounds face major limitations including proteolytic instability, lack of systemic applicability, and poor production yields [42]. Broader activity spectrum, improved delivery systems, and resistance evaluation are crucial for advancing these molecules into clinical use.

4.1. Bacteriocins or AMPs from Clostridium Species

Various antimicrobials produced by the member of the genus Clostridium (

Table 2. Antimicrobial compounds identified from various Clostridium species, along with their producer organism, compound type, molecular characteristics, target spectrum (NR = not reported).

S. No	Compound	Source	Class	Mol. Weight	Mode of Action	MIC Values	Activity Spectrum	Reference
1	Bacteriocin	C. acetobutylicum	Bacteriocin	Sedimentation coefficient is 6S (Sucrose gradient method)	Bactericidal not bacteriolytic	NR	Narrow spectrum (C. acetobutylicum and C. felsineum)	[43]
2	Bacteriocin 28	C. perfringens	Bacteriocin	~100 kDa	NR	NR	Narrow spectrum (Antibacterial)	[44]
3	Bacteriocin N5	C. perfringens	Bacteriocin	82 kDa	Bactericidal	NR	Narrow spectrum (Clostridium strains)	[45]
4	Boticin B	C. botulinum	Bacteriocin	5.1 3 kDa or 4.0 kDa	Bactericidal	NR	Narrow spectrum (C. botulinum strains and related Clostridia)	[46]
5	Boticin P	C. botulinum	Bacteriocin	NR	NR	NR	Narrow spectrum (C. botulinum)	[47]
6	CBP22	C. butyricum	Bacteriocin	2.2 kDa	LPS	128 µg/mL, 64 µg/mL for E. coli ATCC 25922, S. aureus ATCC 26923	Broad spectrum (E. coli ATCC 25922, and S. aureus ATCC 26923)	[48]
7	Circularin and Closticin 574	C. beijerinckii & C. tyrobutyricum	Cyclic antibacterial peptide & Class II Bacteriocin	6.771 kDa and ~2.2 kDa	NR	NR	Narrow spectrum (C. tyrobutyricum, Lactococcus and Lactobacillus strains)	[31]
8	Closthioamide	C. cellulolyticum	Polythioamide Antibiotic	0.69 kDa	Bactericidal	0.5 µg/mL for MRSA, VRE & 9.0 µg/mL for E. coli	Broad spectrum (E. faecalis, Neisseria gonorrhoeae, and Staphylococcus aureus and E. coli)	[34]
9	Clostrubin	C. beijerinckii	Polyphenolic polyketide Antibiotic	0.4 kDa	NR	0.12 to 0.97 µΜ	Narrow spectrum (Mycobacterial strains, VRE, and MRSA)	[29]
10	Clostridocins	C. perfringens and C. butyricum	Bacteriocin	32.5 kDa and 76 kDa	Lytic and non-lytic effect (C. pasteurianum)	NR	Narrow spectrum (Clostridium pasteurianum)	[49]
11	Clostrisin and cellulosin	C. cellulovorans.	Bacteriocin	5.8 kDa and 9.1 kDa	Biostatic	5.6 µg/mL and 4.8 µg/mL for S.epidermidis, and P. aeruginosa.	Narrow spectrum (multidrug- resistant S. epidermidis MIQ43 and Pseudomonas aeruginosa PA14)	[50]
12	Clostrocin A, B, C, D, E	Clostridium spp.	Bacteriocin	NR	Bactericidal	NR	Gram positive bacteria	[51]
13	Clostocin O	Clostridium spp.	Bacteriocin	NR	Bactericidal (Cell wall lysis)	NR	Narrow spectrum (closely related bacterial strains)	[52]
14	Clostrocyloin	C. beijerinckii	Bacteriocin	0.25 kDa	NR	NR	Narrow spectrum (Sporobolomyces, M. vaccae, B. subtilis)	[53]
15	Clostrindolin	C. beijerinckii	Pyrone alkaloid	0.24 kDa	NR	~4 µg/mL	Narrow spectrum (M. vaccae)	[38]
16	Diffocins	C. difficile	R type Bacteriocin	~200 kDa	Bactericidal	NR	Narrow spectrum (other strains of the same Species)	[30]
17	Estericin A	C. estertheticum	Class II lanthipeptide	6.69 kDa	Bactericidal	1 µg/mL for C. perfringens	Narrow spectrum (MRSA, C. perfringens, and C.tetani.)	[54]
18	Intestinalin (P30)	C. intestinale	Bacteriocin	0.023 kDa	Cell membrane permeabilization	0.2 µg/mL	Broad spectrum	[55]
19	Neutral Lipopeptide	C. pasteurianum	Lipopeptide	NR	NR	NR	NR	[56]
20	Perfrin	C. perfringens	Bacteriocin	11.5 kDa	Bactericidal activity (Pore formation)	NA	Narrow spectrum (C. perfringens)	[57]

) reveals biotechnological potentials of this genus for biotherapeutics. However, difficulties in cultivating such anaerobes hampered their detailed studies.

4.1.1. Bacteriocin from Clostridium acetobutylicum

Strains of Clostridium, particularly C. acetobutylicum P62, are used for acetone and butanol production and have also been found to produce a bacteriocin during fermentation [58,59]. This bacteriocin, produced in a molasses-based medium via the Weizmann process [43], exhibits a narrow activity spectrum, inhibiting other strains of C. acetobutylicum and Clostridium felsineum. It is thermolabile, extracellular, and active at pH 4–5, with resistance to proteolytic and nucleolytic enzymes, though it can be inactivated by SDS and phenol. This bacteriocin has a bactericidal effect, reducing cell viability without promoting resistant populations. Unlike other Clostridium bacteriocins linked to autolysis or protease activity, this one is not associated with those processes [60], highlighting its potential as a marker for C. acetobutylicum [43].

4.1.2. Bacteriocin N5

Bacteriocin N5, produced by C. perfringens strain BP6K-N5, was initially induced by UV light. It was purified using ammonium sulfate precipitation, ion-exchange chromatography on DEAE cellulose, and size exclusion chromatography with Sephadex. Its homogeneity was confirmed through polyacrylamide gel electrophoresis [61]. Bacteriocin N5 is a simple protein with a molecular weight of approximately 82 kDa and is characterized as a single polypeptide chain confirmed by SDS-PAGE with β-mercaptoethanol. It exhibits antibacterial activity against certain Clostridium strains, although this activity is significantly reduced after 15 min at 50 °C and upon incubation with proteases. This indicates its poor thermal stability but potential applications [45].

4.1.3. Boticin B and P

Boticin B is a heat-stable bacteriocin produced by C. botulinum strain 213B, capable of inhibiting various other C. botulinum strains and related clostridia. The gene encoding boticin B was cloned from an 18.8-kb plasmid and encodes a 50-amino-acid peptide [46]. Boticin P, a bacteriocin produced by a non-toxigenic strain of C. botulinum type E (PM-15), inhibits the growth of C. botulinum strains lacking the botiA gene. Boticin P consists mainly of protein (98.8%) with a small amount of carbohydrate (0.4%) [47], and its molecular weight exceeds 4 × 10⁶ Da. Boticin P has a static effect on vegetative growth and spore outgrowth of sensitive bacterial strains, but does not affect the initial germination events.

4.1.4. CBP22

CBP22, produced by C. butyricum strain ZJU-F1, was extracted and purified through ammonium sulfate fractionation, cation exchange chromatography, affinity chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC) [58]. It has a low molecular weight of 2264.63 Da, consists of 22 amino acids, and has a +1 net charge [48]. CBP22 showed activity against S. aureus ATCC 26923 and E. coli ATCC 25922, and its antagonistic effects on the innate immune response were studied using a mouse model with lipopolysaccharide induction.

4.1.5. Closticin 574 and Circularin A

Two bacteriocins from Clostridium species, closticin 574 and circularin A, were identified. Closticin 574, produced by Clostridium tyrobutyricum ADRIAT 932, has a preproprotein of 309 amino acids, with an active peptide of 82 amino acids formed through posttranslational modifications. It is secreted via a regular pathway and activated at an Asp-Pro site, exhibiting strong antibacterial activity against other C. tyrobutyricum strains [31]. Circularin A, produced by C. beijerinckii ATCC 25752, consists of a core peptide of 72 amino acids that forms a stable circular structure of 69 amino acids after head-to-tail ligation and removal of a three-amino acid leader peptide. These peptides’ unique structures, particularly circularin A’s circular form, highlight their potential for antimicrobial applications and biotechnological development. Its biosynthetic gene cluster (BGC0000488) comprises the cirABCDE operon. cirA encodes the precursor peptide, while cirB and cirC encode membrane proteins. cirD functions as an ATP-binding transport protein, and cirE provides self-immunity. The cluster is tightly organized, with overlapping ORFs typical of circular bacteriocins. The final compound undergoes enzymatic cyclization via peptide bond formation between the N- and C-termini [31].

4.1.6. Clostocin A, B, C, D and E

Clostocins are produced by non-pathogenic Clostridium strains involved in acetone-butanol or isopropanol-butanol fermentation [62]. The researchers revealed lysogenic strains, producing a temperate phage designated as KT, whereas other strains were identified as bacteriocinogenic, producing bacteriocins categorized into five distinct groups viz., clostocin A, B, C, D, and E. Notably, cross-resistance was found between the KT phage and clostocin A, indicating a shared receptor [51]. A single mutation in this receptor can confer resistance [63]. Clostocins A and D are sensitive to proteolytic enzymes and are thermostable, while B and C are thermolabile. Clostocin D inhibits more Clostridium species, while B and C exhibit a broader activity spectrum against Bacillaceae.

4.1.7. Clostrocyloin

Three strains of C. beijerinckii, known for industrial solvent production, were found to produce new congeners of sattazolin and a novel acyloin named clostrocyloin. The sattazolin compounds exhibited antibacterial activity against Mycobacteria and Pseudomonads with minimal cytotoxicity, suggesting potential as antimicrobial agents [64]. In contrast, clostrocyloin demonstrated efficacy against fungi. The biosynthesis of these compounds was investigated using computational and experimental methods., Researchers have identified a thiamine diphosphate-dependent synthase for biosynthesis of sattazolin and an acyloin synthase for clostrocyloin [53]. These discoveries enhance the understanding of secondary metabolite biosynthesis in C. beijerinckii and suggest avenues for developing new antimicrobial drugs.

4.1.8. Diffocins

Diffocins are R-type bacteriocins produced by certain C. difficile strains that closely resemble Myoviridae phage tails and selectively kill other C. difficile strains. These high-molecular-weight structures are induced by the SOS response and use a contractile needle-like mechanism to disrupt membrane potential in target cells. Killing is initiated by a receptor-binding protein that recognizes specific surface receptors [30,65]. The ~20 kb diffocin locus (ORFs CD1359–1376) includes genes encoding structural components such as the sheath, baseplate, and the strain-specific RBP (ORF1374). Though not yet listed in MIBiG, the cluster has been cloned and functionally characterized [30].

4.1.9. Intestinalin (P30)

Intestinalin (P30) is derived from Clostridium intestinale URNW and is based on the LysC enzyme. Unlike LysC, it demonstrates activity against both Gram-positive and Gram-negative bacteria, including pathogens. Intestinalin (P30) disrupts biofilm formation, reducing biomass by for Klebsiella pneumoniae and Staphylococcus pettenkoferi Notably, intestinalin is non-toxic to mammalian cells, likely due to the presence of zwitterionic phospholipids and cholesterol in their membranes that reduce peptide binding. It selectively targets bacterial cells by interacting with negatively charged phospholipids, forming transmembrane pores that disrupt the electrochemical gradient, impairing ATP synthesis and nutrient transport, thus decreasing bacterial viability [55].

4.1.10. Perfrin

Perfrin is produced by a netB-positive strain of C. perfringens, which carries the netB gene encoding a pore-forming toxin linked to necrotic enteritis, a disease affecting poultry, particularly broilers [66]. It inhibits the growth of other C. perfringens strains and consists of an 11.5 kDa C-terminal portion of a 23 kDa novel protein with no known homology to existing bacteriocins. This active portion was cloned into E. coli, demonstrating bioactivity. While perfrin is chromosomally encoded, the netB gene is plasmid-located. Its antimicrobial activity persists across a wide pH range but decreases significantly at high temperatures and in the presence of proteolytic enzymes like trypsin and proteinase K [57]. The bacteriocin is secreted and likely contains a signal sequence in its N-terminal region.

4.1.11. Clostocin O

Clostocin O is produced by non-pathogenic Clostridium species [52]. It exhibited bactericidal effects against closely related bacterial strains. The mechanism of action of clostocin O showed adsorption to receptor sites on the cell walls of sensitive bacteria, and this adhesion was essential for antibacterial activity.

4.2. Bacteriocins or AMPs from Other Anaerobic Genera

Although the majority of anaerobe-derived antimicrobials are reported by strains of Clostridium species, there are few antimicrobials produced by non-clostridial anaerobes (

Table 3. Antimicrobial compounds produced by non-Clostridium anaerobic genera, along with their producer organism, compound type, molecular characteristics, target spectrum (NR = not reported).

S. No	Compound	Source	Class	Mol. Weight	Mode of Action	MIC Values	Activity Spectrum	References
1	Barnesin	S. barnesii	Lipo-dipeptide	0.48 kDa	Inhibition of cysteine protease	NR	Broad spectrum (Gram positive & Gram negative)	[11]
1	Bifidin I	B. bifidum NCDC 1452	Bacteriocin	2.8 kDa	NR	NR	Broad spectrum (E. coli and S. aureus)	[67]
2	Bifidococcin_664	B. longum subsp. infantis	Bacteriocin	14.9 kDa	NR	NR	Narrow spectrum (C. perfringens)	[68]
3	Naphthalecin	S. colonica	Polyketide antibiotic	0.2 kDa	NR	NR	Gram positive bacteria	[39]
4	Nigrescin	P. nigrescens	Bacteriocin	41 kDa	Bactericidal mode of action	10.7 µg/mL for P. gingivalis	Narrow spectrum (Dental pathogens)	[69]
5	Propionicin T1	P. thoenii	Bacteriocin	7.1 kDa	Bactericidal mode of action	NR	Narrow spectrum (Propionibacterium strains)	[70]
6	Pseudocin 196	B. pseudocatenulatum	Lantibiotic	2.6 kDa	NR	0.2 µm for Lactococcus cremoris HP	Narrow spectrum (Clostridium, Streptococcus, Lactococcus spp.)	[71]
7	Ruminicoccin	R. gnavus	sactipeptide	NR	NR	0.8 to 50 µg/mL	Narrow spectrum (S. aureus VRE, nisin-resistant Bacillus subtilis and methicillin-resistant S. aureus (MRSA)	[72,73]

). Notably, members of the genus like Bifidobacterium, that are considered as probiotics, have emerged as promising producers of AMPs with applications such as food preservation.

4.2.1. Bifidin I

Bifidobacterium bifidum NCDC 1452, is a probiotic strain that produces bifidin, effective against E. coli and S. aureus [74]. Bifidin I, also produced by Bifidobacterium infantis BCRC 14602 [67], is purified through adsorption-desorption on silicic acid, cation-exchange chromatography at pH 7.6, and reverse phase high-performance liquid chromatography (RP-HPLC). It has a molecular weight of 2.8 kDa, determined by MALDI-TOF. Bifidin I shares homology with pediocin PA1-like Class IIa bacteriocins, featuring a -Tyr-Gly-Asn-Gly-Val-Xaa-Cys (YGNGV) consensus sequence, known for their strong antilisterial activity [75,76]. This strain is valuable in the dairy industry and food preservation, as bifidin I is plasmid encoded, with Bif¯ variants failing to secrete it [67].

4.2.2. Bifidococcin_664

Bifidococcin_664, is produced by Bifidobacterium longum subsp. infantis LH_664 [50]. It has a molecular weight of 14.9 kDa and consists of 134 residues, including a 34-amino acid leader sequence at the N-terminus (MSVQTGK) and a C-terminus (AY YYRVS). With 60% sequence homology to other known bacteriocins, Bifidococcin_664 exhibited 19 narrow-spectrum activity against C. perfringens LH [68]. Additionally, it induces NF-κB activation and IL-8 release in THP1-Blue monocytes, suggesting a role in modulating the host immune response.

4.2.3. Nigrescin

Nigrescin is produced by the anaerobic strain Prevotella nigrescens ATCC 25261, encoded by the nig locus with parallel genes nigA–D. With a molecular weight of approximately 41 kDa, Nigrescin is significantly larger than typical AMPs [69]. It exhibits a bactericidal mode of action against dental pathogens such as Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Actinomyces species, while sparing commensal bacteria like Lactobacillus and Streptococcus. Nigrescin is heat-resistant but sensitive to proteinase K, making it suitable for use in pasteurized products. It is non-toxic to periodontal cell linings and active within a pH range of 6.5 to 9.5, positioning it as a potential therapeutic agent for periodontal disease prevention and treatment.

4.2.4. Propionicin T1

Propionicin T1 is produced by Propionibacterium thoenii [70]. It shows no sequence homology with other known bacteriocins and has antimicrobial activity against P. thoenii, Propionibacterium acidipropionici, and Propionibacterium jensenii, but not against Propionibacterium freudenreichii. This specificity makes it a potential agent in cheese production to prevent contamination from certain strains while protecting the starter culture [77]. It is synthesized as a 96-amino acid prepeptide with a sec leader, becoming a mature 65-amino acid bacteriocin afterward. It is a small, cationic peptide that is thermostable, retaining activity after freezing, thawing, and even after six months of storage at various temperatures. However, treatment with proteinase K inactivates its antimicrobial activity. Additionally, a novel E. coli–Propionibacterium shuttle vector was developed for efficient transformation of P. freudenreichii, enabling the cloning and expression of propionicin T1 and pro-PAMP [77]. This genetic system supports functional gene studies and development of enhanced strains.

5. Thiopeptides and Related RiPPs from Anaerobes

Thiopeptides and lanthipeptides are subclasses of ribosomally synthesized and post-translationally modified peptides (RiPPs), known for their complex structures and potent antimicrobial activity. While thiopeptides feature heterocyclic cores rich in thiazole and oxazole rings, lanthipeptides are defined by thioether cross-links and remain one of the most studied RiPP classes. Lanthipeptides represent a prominent subclass of RiPPs, distinguished by the presence of characteristic thioether cross links lanthionine and methyllanthionine formed through the enzymatic dehydration of serine/threonine residues followed by addition of cysteines [78]. Genomic analyses have revealed diverse lanthipeptide BGCs across multiple bacterial phyla, significantly expanding the known chemical diversity beyond traditional producers. The polycyclic structures confer high conformational stability and resistance to proteolysis, often resulting in potent antimicrobial activities, lantibiotics being the classical example. Their high specificity and low toxicity toward mammalian cells make them attractive candidates for therapeutic development. Moreover, their biosynthetic pathways are genetically tractable, allowing for bioengineering of novel analogs [78]. However, lanthipeptides also face limitations such as poor solubility, variable stability at physiological pH, low native production yields [79]. Addressing these challenges is essential to unlocking their clinical potential.

5.1. Clostrisin and Cellulosin

In silico genome mining of Clostridium cellulovorans 743B identified nine BGCs, including a super-cluster with two novel lanthipeptides, clostrisin and cellulosin. Clostrisin and cellulosin were expressed in E. coli IM08B, purified, and tested for bioactivity. Mass spectrometry confirmed post-translational modifications in cellulosin, while clostrisin remained unmodified [50]. Both displayed antimicrobial activity against multidrug-resistant S. epidermidis MIQ43 and Pseudomonas aeruginosa PA14 but were ineffective against E. coli IM08B and C. difficile. Notably, cellulosin contains large thioether rings, whereas clostrisin lacks any dehydrated residues or lanthionine rings.

5.2. Estericin A

Estericin A is a class II lanthipeptide encoded by the BBGC7 cluster on a ~180 kb plasmid in Clostridium estertheticum CF016 (MIBiG: BGC0002667). The cluster includes structural genes, a radical SAM enzyme, ABC transporters, immunity proteins, and proteases. Genome mining and bioactivity assays confirmed its biosynthesis and antimicrobial potential [33]. Estericin A was successfully cloned and expressed in E. coli [54], demonstrating activity against MRSA, C. perfringens, and Clostridium tetani. It inhibits cell wall synthesis by binding lipid II, without pore formation. The 27-amino acid peptide contains three (methyl)lanthionine rings, conferring stability across a broad pH range and temperatures up to 95 °C, though it remains sensitive to proteases. Its BGC organization and predicted structure (Figure 3) illustrate typical class II lanthipeptide features, including thioether cross-links.

5.3. Pseudocin 196

Bifidobacterium pseudocatenulatum MM0196, a faecal isolate from a healthy pregnant woman, produced a pseudocin 196 [80,81]. It has a molecular weight of 2679 Da and is similar to lacticin 481 from Lactococcus lactis. It exhibits antibacterial activity against Clostridium and Streptococcus sp. Its low MIC values and broad inhibitory spectrum highlight its therapeutic potential. The discovery of pseudocin 196 suggests that B. pseudocatenulatum MM0196 may serve as a beneficial probiotic [71], but further research is needed to understand its mechanism of action and clinical efficacy.

5.4. Ruminococcin

Like lanthipeptides, sactipeptides are RiPPs that gain structural stability through the formation of thioether cross-links through sulfur-to-α-carbon (sactionine) bonds rather than lanthionine bridges. Sactipeptides undergo posttranslational modifications, and their biosynthetic clusters encode enzymes for dehydration and thioether bond formation (Figure 4). Ruminococcin A and ruminococcin C were reported from strains of Ruminococcus gnavus [72,73]. Ruminococcin C is a sactipeptide produced by the gut isolate Ruminococcus gnavus strain E1, marking the first bacteriocin reported from the Ruminococcus genus. The BGC (BGC0002043) spans 12.8 kb and includes 13 genes organized in three operons. Notably, it encodes three nearly identical structural genes (rumA1–3), a radical SAM enzyme (rumM), an ABC transporter (rumT), and regulatory proteins. Transcription is strongly induced by trypsin, indicating a growth phase- and protease-dependent regulatory mechanism. This regulation is mediated by a two-component system encoded within the same region [32]. Ruminococcin is effective against Clostridium pathogens, including C. difficile, and was found to be non-toxic to intestinal and gastric cell lines. An analogous biosynthetic cluster, RumC, was found to encode a sactipeptide with four thioether bridges, contributing to its stability under extreme conditions [82,83]. Moreover, R. gnavus E1 may positively influence gut homeostasis by increasing butyrate and acetate levels and decreasing propionate and ammonia [32].

6. Glycopeptides from Anaerobes

Glycopeptides are a class of nonribosomally synthesized antibiotics characterized by a heptapeptide scaffold modified with aromatic cross-links and sugar moieties, which enable high-affinity binding to lipid II and inhibition of peptidoglycan synthesis [84]. Classical glycopeptides such as vancomycin and teicoplanin have been pivotal in treating Gram-positive infections. Anaerobe-derived glycopeptides mirror this chemical complexity and hold potential for combating resistant pathogens. However, as with their aerobic counterparts, their therapeutic development is limited by several factors: high molecular weight and poor solubility reduce tissue penetration, while biosynthetic complexity makes scalable production challenging [85]. Recent research underscores the importance of tailoring glycopeptide delivery using strategies such as liposomal encapsulation, PEGylation, or prodrug design to overcome these barriers and improve systemic bioavailability [86,87]. Advancing our understanding of their biosynthetic gene clusters and structure–activity relationships will be critical to unlocking their translational potential.

6.1. Clostridocins

Two bacteriocins, butyricin 7423 and perfringocin 11105, were isolated from Clostridium butyricum NCIB 7423 and Clostridium perfringens type A NCIB 11105. They exhibited different modes of action on Clostridium pasteurianum and were amphiphilic, with sensitivity to trypsin. Both could bind to non-ionic detergents like Triton X-100, with butyricin 7423 acting as a hydrophobic protein in its presence. Purification of butyricin 7423 involved Triton-X binding, ammonium sulfate precipitation, and gel filtration through Sepharose 6B, followed by chromatography on Sephadex LH-20 to remove Triton X-100. Although samples showed variable carbohydrate content, bioactivity persisted without it. Butyricin 7423 had a molecular weight of 32.5 kDa, while perfringocin 11105 was a single protein with a molecular weight of 76 kDa [49].

6.2. Bacteriocin 28

Bacteriocin 28, produced by C. perfringens, identified as a glycoprotein with a molecular weight of 100 kDa via gel filtration and 84 kDa through density gradient centrifugation. It exhibited hydrophobic properties, strongly bound to phenyl-Sepharose CL-4B gel, and was eluted with ethylene glycol [44]. It was immunogenic, producing precipitating and neutralizing antibodies, and contains 15 amino acids and carbohydrate moieties.

7. Lipopeptides from Anaerobes

Lipopeptides are amphiphilic antimicrobial compounds composed of a cyclic or linear peptide backbone covalently linked to one or more fatty acid chains, enabling interaction with bacterial membranes [88]. Their mechanism of action typically involves insertion into the lipid bilayer, leading to membrane disruption, ion leakage, and rapid bactericidal activity. Clinically approved lipopeptides such as daptomycin have proven highly effective against multidrug-resistant Gram-positive pathogens, including MRSA and VRE [89]. Lipopeptides exhibit potent, broad-spectrum membrane-disrupting activity. However, their clinical translation is hindered by notable limitations like high hydrophobicity contributing to poor aqueous solubility and formulation challenges; nonspecific membrane targeting resulting in hemolytic activity and cytotoxicity [89]. To address these obstacles, future strategies must focus on, engineering analogs with improved selectivity and reduced toxicity, and developing delivery systems that enhance bioavailability under physiological conditions.

7.1. Barnesin A

Barnesin A was a hybrid nonribosomal peptide-polyketide metabolite produced anoxically by Sulfurospirillum barnesii (Epsilon-proteobacterium). Its biosynthetic gene cluster (BGC0001524) spans approximately 20–30 kb and encodes both NRPS and PKS modules, as well as regulatory and tailoring enzymes. The genes were arranged in a colinear modular fashion, and the expression of the cluster has been confirmed via transcriptomic analysis [11] This rare compound, consisting of vinylogous amino acids, is produced by a BGC (Figure 5) that includes nine genes (BrnA−BrnI) responsible for key enzymes in NRPS and PKS pathways, along with a phosphopantetheinyl transferase and a cyclic peptide transporter [11]. The presence of conserved 24 bp and 12 bp direct repeats indicates horizontal gene transfer. Barnesin A features an amphiphilic lipopeptide structure and demonstrates potent activity against a broad spectrum of microbes [11]. It showed moderate to strong antibacterial activity against S. aureus, Mycobacterium vaccae, and P. aeruginosa, making it a promising candidate for antimicrobial drug deveIts significance lies in being one of the few anaerobically synthesized secondary metabolites with notable bioactivity.

7.2. Neutral Lipopeptide

C. pasteurianum produces surface active compounds (SACs) in anaerobic sucrose medium, including lipids with both hydrophilic and hydrophobic components. The fermentation lowered broth surface tension, leading to the production of neutral lipopeptides [56], which were isolated from the cultured supernatant using foam fractionation. Analysis revealed one of the lipids as a triglyceride and two others as possible esters. However, limited information is available regarding this neutral lipopeptide.

8. Translational Challenges in Clinical Development of Anaerobic-Derived Antimicrobials

Despite the promising diversity and bioactivity of antimicrobial compounds derived from anaerobic microorganisms, their translation from bench to bedside faces several challenges. One major barrier is the lack of regulatory precedents for drugs developed from strict anaerobes. Regulatory agencies such as the Food and Drug Administration or European Medicines Agency often require extensive safety, toxicity, and pharmacokinetics data, which are difficult to obtain for compounds produced by organisms that are difficult to culture, genetically manipulate, or scale up under industrial conditions [27].

Additionally, few anaerobe-derived antimicrobials have advanced into clinical trials, largely due to limited investment and the absence of preclinical models specifically designed for anaerobic drug discovery. For example, bacteriocins such as Nisin, produced by Lactococcus lactis, a facultative anaerobe, have been granted GRAS (Generally Recognized As Safe) status for food preservation. Several natural and bioengineered variants of nisin, including nisin A, Z, Q, and H, have shown promising antimicrobial activity in preclinical studies against drug-resistant clinically relevant pathogens [79]. However, none have progressed to clinical trials for therapeutic use, primarily due to challenges related to compound stability, delivery, and immunogenicity. Efforts to translate these findings into clinical applications have largely been unsuccessful, hindered by limited efficacy compared to standard-of-care treatments, suboptimal clinical trial designs, and manufacturing constraints. Similarly, several potent compounds from obligate anaerobes such as closthioamide, clostrubin, and CBP22 have demonstrated significant in vitro activity but remain at the preclinical stage, with no advancement to animal models or human trials to date.

Manufacturing and formulation challenges further hinder commercialization. Many compounds from anaerobes require anaerobic fermentation conditions or host organisms not amenable to large-scale production [14]. Moreover, complex structures (e.g., lanthipeptides or thioamide-containing molecules) may demand synthetic or semi-synthetic strategies for consistent drug development, which increases cost and complexity. Another reason many natural product-encoding genes remain untapped is that they are often transcriptionally repressed under native conditions; as a result, numerous bioactive molecules are likely overlooked in standard cultivation or screening protocols [15]. Finally, intellectual property and market incentives are also limiting factors. Since antimicrobials are used sparingly to reduce resistance development, commercial returns are often lower than for chronic-use drugs, reducing industry interest [27]. This is especially true for niche products targeting multidrug-resistant pathogens or specific anaerobic infections.

Addressing these translational hurdles will require interdisciplinary collaborations between microbiologists, chemists, pharmaceutical scientists, and regulatory experts, as well as policy reforms to incentivize antimicrobial development from novel sources like anaerobes.

9. Conclusions and Future Research Directions

Anaerobic microorganisms offer a vast but underutilized reservoir of antimicrobial compounds. While recent genome mining has uncovered diverse biosynthetic gene clusters, major advances are needed to translate these discoveries into clinical candidates. Priority areas include expanding cultivation methods to access unculturable strains and using advanced bioinformatics to prioritize novel BGCs.

Developing anaerobic or facultative expression platforms will be essential for scalable production, alongside omics-driven strategies to link genotype to metabolite output. Establishing in vivo models that mimic anaerobic infection sites will support efficacy testing. Synthetic biology and pathway engineering can improve yields and diversify compound structures.

Progress will also depend on policy support and targeted funding to overcome commercial and regulatory barriers. With a coordinated effort, anaerobe-derived antimicrobials can emerge as a vital component of the global response to antimicrobial resistance.