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Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes listeriosis, a serious foodborne illness. In the nature-to-human transmission route, Lm can prosper in various ecological niches. Soil and decaying organic matter are its primary reservoirs. Certain clonal complexes (CCs) are over-represented in food production and represent a challenge to food safety. To gain new understanding of Lm adaptation mechanisms in food, the genetic background of strains found in animals and environment should be investigated in comparison to that of food strains. Twenty-one partners, including food, environment, veterinary and public health laboratories, constructed a dataset of 1484 genomes originating from Lm strains collected in 19 European countries. This dataset encompasses a large number of CCs occurring worldwide, covers many diverse habitats and is balanced between ecological compartments and geographic regions. The dataset presented here will contribute to improve our understanding of Lm ecology and should aid in the surveillance of Lm. This dataset provides a basis for the discovery of the genetic traits underlying Lm adaptation to different ecological niches.

In this contribution, the antimicrobial susceptibility toward 11 antibiotics and four biocides of a panel of 205 Listeria monocytogenes (Lm) strains isolated from different ecological niches (i.e., food, animals and natural environment) was evaluated. The impact of exposure to biocides on the antibiotic susceptibilities of Lm was also investigated. Lm strains isolated from food exhib-ited overall a lower susceptibility (higher minimal inhibitory concentrations, MIC) for ammonium quaternary compounds (QACs) and peracetic acid (PAC) than strains isolated from animals and natural environments. Conversely, the ecological origins of Lm strains did not significantly affect their susceptibilities towards antibiotics. Interestingly, repeated exposure to QACs recurrently led to a decrease in susceptibility toward ciprofloxacin (CIP), a fluoroquinolone antibiotic, largely used in human medicine. Moreover, these lower levels of susceptibility to CIP remained stable in most Lm strains even after subcultures without biocide selection pressure, suggesting an adaptation involving modifications at the genetic level. Results underlined the ability of Lm to adapt to biocides, especially QACs, and the potential link between this adaptation and the selection of resistance toward critical antibiotics such as ciprofloxacin. These data support a potential role of the extensive use of QACs from “farm to fork” in the selection of biocide and antibiotic resistance in pathogenic bacteria such as Lm.

International audience

A workshop on the risks associated with animal botulism was held in Maisons-Alfort, France in March 2019. The objectives were to debate the public and animal health risks related to botulism, to disseminate the results obtained from the ANIBOTNET project to other European research teams that were not involved in the project and to other stakeholders, and finally to strengthen and broaden the existing research network on this topic. The workshop was divided into 4 sessions: public and animal health risks associated with botulism, a specific focus on risks associated with agricultural biogas plants, dissemination of ANIBOTNET results, and network development and future perspectives. In all, 58 delegates from 13 countries attended the workshop. A summary of the main topics and discussions during the workshop is presented here.

Botulism is a statutory notifiable disease in Portugal since 1999, with 112 cases registered until 2015. The largest number of cases reported per year was 30 cases, from 2 outbreaks in 2000. Most foodborne cases were caused by botulinum neurotoxin type B found in home preserved meat products.The first infant botulism case occurred in 2012. Only one case of type F toxin was ever reported in Portugal. N/A

Botulinum neurotoxins are diverse proteins. They are currently represented by at least seven serotypes and more than 40 subtypes. New clostridial strains that produce novel neurotoxin variants are being identified with increasing frequency, which presents challenges when organizing the nomenclature surrounding these neurotoxins. Worldwide, researchers are faced with the possibility that toxins having identical sequences may be given different designations or novel toxins having unique sequences may be given the same designations on publication. In order to minimize these problems, an ad hoc committee consisting of over 20 researchers in the field of botulinum neurotoxin research was convened to discuss the clarification of the issues involved in botulinum neurotoxin nomenclature. This publication presents a historical overview of the issues and provides guidelines for botulinum neurotoxin subtype nomenclature in the future.

Clostridium botulinum produces the most potent natural toxin, the botulinum neurotoxin (BoNT), probably to create anaerobiosis and nutrients by killing the host, and forms endospores that facilitate survival in harsh conditions and transmission. Peak BoNT production coincides with initiation of sporulation in C. botulinum cultures, which suggests common regulation. Here, we show that Spo0A, the master regulator of sporulation, positively regulates BoNT production. Insertional inactivation of spo0A in C. botulinum type E strain Beluga resulted in significantly reduced BoNT production and in abolished or highly reduced sporulation in relation to wild-type controls. Complementation with spo0A restored BoNT production and sporulation. Recombinant DNA-binding domain of Spo0A directly bound to a putative Spo0A-binding box (CTTCGAA) within the BoNT/E operon promoter, demonstrating direct regulation. Spo0A is the first neurotoxin regulator reported in C. botulinum type E. Unlike other C. botulinum strains that are terrestrial and employ the alternative sigma factor BotR in directing BoNT expression, C. botulinum type E strains are adapted to aquatic ecosystems, possess distinct epidemiology and lack BotR. Our results provide fundamental new knowledge on the genetic control of BoNT production and demonstrate common regulation of BoNT production and sporulation, providing a key intervention point for control.

Bacteria have evolved a number of mechanisms for coping with stress and adapting to changing environmental conditions. Many bacteria produce small cold shock proteins (Csp) as a response to rapid temperature downshift (cold shock). During cold shock, the cell membrane fluidity and enzyme activity decrease, and the efficiency of transcription and translation is reduced due to stabilization of nucleic acid secondary structures. Moreover, protein folding is inefficient and ribosome function is hampered. Csps are thought to counteract these harmful effects by serving as nucleic acid chaperons that may prevent the formation of secondary structures in mRNA at low temperature and thus facilitate the initiation of translation. However, some Csps are non-cold inducible and they are reported to be involved in various cellular processes to promote normal growth and stress adaptation responses. Csps have been shown to contribute to osmotic, oxidative, starvation, pH and ethanol stress tolerance as well as to host cell invasion. Therefore, Csps seem to have a wider role in stress tolerance of bacteria than previously assumed. Yersinia enterocolitica and Yersinia pseudotuberculosis are enteropathogens that can spread through foodstuffs and cause an enteric infection called yersiniosis. Enteropathogenic Yersinia are psychrotrophs that are able to grow at temperatures close to 0 degrees C and thus they set great challenges for the modern food industry. To be able to efficiently control psychrotrophic Yersinia during food production and storage, it is essential to understand the functions and roles of Csps in stress response of enteropathogenic Yersinia.

Core genome phylogenies of Group I strains. A) Core genome phylogeny of C. botulinum Group I strains inferred with RAxML [63] using the ASC_GTRGAMMA model on an alignment of 182,200 core genome SNPs produced with NASP [61] using C. sporogenes ATCC 15579 [GenBank:ABKW00000000] as the reference genome. The consistency index is 0.56, and the retention index is 0.91 (computed with the R package phangorn). B) Core genome phylogeny of Group I strains inferred with FastTree2 [54] on a 1780-character core SNP matrix generated with kSNP [59]. The consistency index is 0.64, and the retention index is 0.93. The phylogenies were rooted with the clade that includes C. sporogenes and C. botulinum bont/B serotypes (bottom of Figure). Gray circles indicate bootstrap values over 95 %. While there are small variations in the phylogenies generated with different methods (Fig. 5 and Additional file 4: Figure S1), the overall topology of the Group I tree appears robust. The pairwise overall topological scores computed by Compare2Trees [69] range from 80 to 86 % for the phylogenies presented in Fig. 5 and (Additional file 4: Figure S1). (PDF 59 kb)

Core genome phylogenies of Group II strains. A) Core genome phylogeny of C. botulinum Group II strains inferred with RAxML [63] using the ASC_GTRGAMMA model on an alignment of 200,276 core genome SNPs produced with NASP [61] using C. botulinum strain Alaska E43 [GenBank:CP001078] as the reference. The consistency index is 0.81, and the retention index is 0.90 (computed with the R package phangorn). B) Core genome phylogeny of Group II C. botulinum strains inferred with FastTree2 [54] on a 35,382-character core SNP matrix generated with kSNP [59]. The consistency index is 0.83, and the retention index is 0.90. The phylogenies were rooted with the clade that includes strains Eklund 202F, KAPB-3, Eklund 17B and CDC 66177. Gray circles indicate bootstrap values over 95 %. While there are small variations in the phylogenies generated with different methods (Fig. 6 and Additional file 6: Figure S3), Group II strains are separated into two distinct clades. The pairwise overall topological scores computed by Compare2Trees [69] range from 93 to 100 %. (PDF 35 kb)