Overview of botulinum neurotoxin-producing clostridia in soils in France

Abstract

Soil is a known reservoir of human pathogens, including botulinum neurotoxin-producing clostridia. However, the occurrence and distribution of botulinum neurotoxin types in French soils are unknown, prompting this study. Botulinum neurotoxin-producing clostridia were sought, and botulinum neurotoxin types were identified through an analysis of 486 soil samples representative of nationwide soil type and land use, provided by the French Soil Quality Monitoring Network. Botulinum neurotoxin-producing clostridia were detected in 45.3% of soil samples, with type B-producing clostridia being the most frequently detected (40.1% of soil samples). Geographical variability was observed, with samples from the North of France being more frequently positive than those from the South. A comparison of these detection results with the characteristics of soils and climate conditions revealed three soil clusters. Clostridium botulinum type A was overrepresented in the first cluster, comprising sandy soils, with a higher C:N ratio and higher yearly precipitation than in all the other soil samples. Type B-producing clostridia were overrepresented in the other two clusters, comprising silty soils in cluster 2 and clay soils in cluster 3 with a high concentration of cationic minerals and a higher cation exchange capacity and pH than the average among all samples. Finally, a non-toxic surrogate strain was seen to have survived and was monitored in different selected soils under various laboratory conditions. It remained highly stable, with persistent spores.

Importance: Botulism is a flaccid paralysis disease caused by one of the seven neuroparalytic toxins produced by anaerobic spore-forming clostridia. While soil is reported in the literature as being an important reservoir of clostridia capable of producing botulinum neurotoxins, no study has been conducted in France up to now to establish its prevalence and study soil factors influencing its occurrence. The significance of our research is in providing a global picture of the unequal distribution of botulinum neurotoxin-producing clostridia and detected subtypes countrywide, showing that types involved in human botulism outbreaks are commonly detected, while those involved in animal outbreaks are rare. Finally, the stability of spores was evaluated. Results showed a high persistence of spores under tested conditions. This study provides new data regarding the distribution of botulinum neurotoxin-producing clostridia in soils that are crucial for a better understanding and management of animal and human botulism outbreaks.

Keywords: Clostridium botulinum; botulinum neurotoxin; clostridia; soil.

Fig 1

Geographical distributions of soil samples according to (A) frequency (%) of detection of BoNT-producing clostridia per department and (B) types of BoNT-producing clostridia in France (471 soil samples, 2020–2023). Maps are from GEOFLA.

Fig 2

Principal component analysis (PCA) of soil characteristics and climate conditions (A) and characteristics of soil sample clusters obtained from hierarchical ascendant classification of principal components (B) (471 soil samples, France, 2020–2023). Results of the PCA are shown as contributions (contrib) of the variables to the first two dimensions of the PCA.

Fig 3

Survival of C. novyi in microcosms of four contrasting soils—A, B, C, and D—after 6 months of incubation under aerobic conditions at 20°C. A bilateral Kruskal-Wallis test was used to compare the distributions of spore numbers, and a bilateral Dunn test was used to estimate P values.

Fig 4

C. novyi spore counts in soil microcosms according to incubation time. Counts were pooled for soils A, B, C, and D at each incubation period, as depicted in Table 3. Soil microcosms were incubated at 20°C under aerobic conditions. A bilateral Kruskal-Wallis test was used to compare the numbers of spores along time, and a bilateral Dunn test was used to estimate P values.