Botulism is a potentially fatal neurotoxin-mediated disease characterized by a symmetrical, descending flaccid paralysis affecting voluntary and autonomic muscles. Many patients develop respiratory compromise, frequently requiring emergent airway support, intensive care, and antitoxin administration. Different forms are distinguished by the route of the toxin entry, such as foodborne botulism (FB), intestinal or infant botulism (IB), as well as wound, inhalational, and iatrogenic (cosmetic or therapeutic) botulisms (Rao et al., 2021). The botulinum neurotoxin (BoNT) is one of the most lethal known substances, produced by seven distinct Clostridium species: Clostridium parabotulinum, Clostridium sporogenes, Clostridium botulinum, Clostridium novyi sensu lato, Clostridium argentinense, Clostridium butyricum, and Clostridium baratii. But, over the latter half of the 20th century, the use of C. botulinum to identify any BoNT-producing bacterium gained widespread acceptance (Smith et al., 2023). Based on amino acid variations and antigenic activity, eight toxin types (BoNT/A-BoNT/H) and at least 41 subtypes have been identified in clusters with ha or orfX conformation (Hill et al., 2007).

Laboratory diagnostic criteria for botulism are based on the isolation of BoNT-producing clostridia, the detection of BoNT, or the identification of their encoding genes (bont) https://www.ecdc.europa.eu/en/botulism/facts. A variety of technologies and their derivatives—including cell-based assays, endopeptidase mass spectrometry (Endopep-MS), immunoassays, nucleic acid testing, comparative genomic methods, and electrochemical biosensors—have been developed to detect and characterize the causative agents of botulism (Centurioni et al., 2022; Wang et al., 2025). Diagnosis mainly relies on the mouse bioassay (SMB) and the bont gene amplification (Lindström et al., 2001; Pillai et al., 2024). Diagnosis methods possess serious drawbacks. Specifically, SMB requires animal facilities and the bont type-specific PCR assays are limited by sequence diversity within subtypes (≥23.7%) (Peck et al., 2017; Pillai et al., 2024). Single nucleotide polymorphisms can affect primer or probe matches, potentially resulting in detection failure (Pillai et al., 2024).

Difficulties in detecting BoNT may be mitigated by identifying the nontoxin-nonhemagglutinin (ntnh) gene using a single PCR assay (Raphael and Andreadis, 2007; Fach et al., 2009; Hill et al., 2010; Williamson et al., 2017). All BoNTs are naturally co-expressed with a protective partner, the NTNH protein, forming together the minimal progenitor toxin complex (Lam and Jin, 2015). The role of NTNH in the disease is crucial, as it stabilizes and protects BoNT from the acidic environment and proteases present in the host’s gastrointestinal tract (Gao and Jin, 2024). The ntnh gene is highly conserved and is associated with the BoNT complex across all types, subtypes, and variants, but is absent in non-toxigenic strains (Raphael and Andreadis, 2007; Fach et al., 2009; Hill et al., 2010; Williamson et al., 2017). This study evaluates ntnh gene detection as a strategy to enhance the molecular diagnosis of botulism.

The effectiveness of molecular tests in reliably detecting all known BoNT subtype variants causing botulism is constrained by the genetic diversity of BoNTs, even within toxin types (Hill et al., 2010; Peck et al., 2017). To address this constraint on accuracy, the detection of NTNH offers an alternative. The ntnh gene was exclusively found in toxin-producing clostridia and displayed a high co-presence pattern with bont gene. This confirms that the heteromeric stabilized neurotoxin is the actual virulence factor and that ntnh-based diagnostic methods are therefore causal. Despite the ntnh gene is a known “hotspot” for recombination, insertions, and mosaicism (East et al., 1996; Harris et al., 2024), it was found more conserved than BoNT variants and is thus amenable to widespread detection less multiplexing PCR.

C. botulinum has a relatively stable genome except for the bont gene cluster where ntnh gene is included (Hill et al., 2009). Most of these clusters are flanked by insertion sequence elements encoding transposases that may be capable of horizontal gene transfer between C. botulinum Groups I and II, or between species (Smith et al., 2015). Regarding the variability of ntnh and bont genes across different strains, we observed that NTNH sequences within the same BoNT subtype exhibit a high degree of redundancy. Our results show that within intra-subtype comparisons (representing short-term evolution), BoNT is marginally more conserved than NTNH (99.8 ± 0.3% vs. 97.1 ± 5.7% identity), which is expected as the BoNT variant defines the subtype itself. However, this trend reverses during mid-term evolution (different subtypes within the same type), where NTNH shows higher conservation (90.0 ± 10.6% vs. 88.3 ± 11.9%). This conservation gap is greatly enhanced in long-term evolution (different toxin types), where NTNH identity remains significantly higher than BoNT (67.2 ± 8.9% vs. 39.7 ± 8.4%).

NTNH plays a conserved core role in stabilizing and protecting BoNTs across their diverse natural environments (soil, decaying matter, etc.), forming a stable and large toxin complex (Lam and Jin, 2015) that prevents the toxin from being broken down by enzymes (like proteases) before it can infect a host. However, NTNH is also highly flexible, customizing its structure and function to adapt to either the ntnh-ha cluster or the orfX-ntnh cluster (a smaller complex). This suggests that NTNH provides an evolutionarily flexible platform for developing new BoNT variants, enabling it to fit into different environments and attack specific host organisms and tissues. The adaptability of NTNH is thus tied directly to the evolutionary progress of BoNTs (Gao and Jin, 2024; Pitel et al., 2025). Consequently, the NTNH protein acts as an “unsung hero” that not only protects the toxin but actively contributes to its evolutionary success and specialization.

While a wide spectrum of PCR assays has been developed to detect bont genes, only a limited number of studies have incorporated the ntnh gene (Raphael and Andreadis, 2007; Fach et al., 2009; Hill et al., 2010; Williamson et al., 2017). This limited implementation may be due to the fact that ntnh detection does not inherently identify the specific toxin serotype. Previous studies have integrated ntnh gene into various platforms, including real-time PCR (Raphael and Andreadis, 2007; Fach et al., 2009), quantitative PCR (Hill et al., 2010), and conventional PCR (Williamson et al., 2017). The assay designed by Williamson et al. was selected because it also provides epidemiological information regarding the neurotoxin cluster type and has been validated across C. botulinum Group I, C. sporogenes, and C. botulinum Group II (Williamson et al., 2017).

In botulism diagnostics, discrepancies between the detection of BoNT (the protein) and its corresponding genes (bont) are a well-documented event (Centurioni et al., 2022). Here, the application of ntnh detection in diagnosis proved to be a useful complementary target for rapidly confirming positive results from the SMB or bont PCR (26 cases, 35.62%). High correlation was found between the presence of NTNH and BoNT (96.5% of the analyzed genomes).

A potential limitation of ntnh detection is non-expressed toxin genes (Raphael and Andreadis, 2007), although this event does not occur in our study, with the detection of ntnh-ha and ntnh-orfX clusters in a BoNT/A1(B5) case. Furthermore, the ntnh assay provides only indirect evidence for BoNT presence and relies on the functional assumption that BoNT and NTNH are mutually required. While the current criteria for laboratory confirmation of botulism requires explicit positive results from the SMB and/or bont gene identification, there are no established guidelines for interpreting a positive ntnh gene detection when both the SMB and bont tests are negative. This gap in guidelines is highlighted by its occurrence in two patients within BoNT- diagnostic group who were later clinically diagnosed with food-borne botulism.

In conclusion, the detection of ntnh represents a valuable addition to our diagnostic arsenal of botulism. Our findings suggest the need for establishing guidelines on how to interpret a positive ntnh result in the absence of direct toxin and bont gene confirmation to maximize the potential of this target.