Strategies for developing phages into novel antimicrobial tailocins.

Tailocins are high-molecular-weight bacteriocins produced by bacteria to kill related environmental competitors by binding and puncturing their target. Tailocins are promising alternative antimicrobials, yet the diversity of naturally occurring tailocins is limited. The structural similarities between phage tails and tailocins advocate using phages as scaffolds for developing new tailocins. This article reviews three strategies for producing tailocins: disrupting the capsid-tail junction of phage particles, blocking capsid assembly during phage propagation, and creating headless phage particles synthetically. Particularly appealing is the production of tailocins through synthetic biology using phages with contractile tails as scaffolds to unlock the antimicrobial potential of tailocins.

A Novel Prophage-like Insertion Element within yabG Triggers Early Entry into Sporulation in Clostridium botulinum.

Sporulation is a finely regulated morphogenetic program important in the ecology and epidemiology of Clostridium botulinum. Exogenous elements disrupting sporulation-associated genes contribute to sporulation regulation and introduce diversity in the generally conserved sporulation programs of endospore formers. We identified a novel prophage-like DNA segment, termed the yin element, inserted within yabG, encoding a sporulation-specific cysteine protease, in an environmental isolate of C. botulinum. Bioinformatic analysis revealed that the genetic structure of the yin element resembles previously reported mobile intervening elements associated with sporulation genes. Within a pure C. botulinum culture, we observed two subpopulations of cells with the yin element either integrated into the yabG locus or excised as a circular DNA molecule. The dynamics between the two observed conformations of the yin element was growth-phase dependent and likely mediated by recombination events. The yin element was not required for sporulation by C. botulinum but triggered an earlier entry into sporulation than in a related isolate lacking this element. So far, the yin element has not been found in any other C. botulinum strains or other endospore-forming species. It remains to be demonstrated what kind of competitive edge it provides for C. botulinum survival and persistence.

Engineering of Salmonella Phages into Novel Antimicrobial Tailocins.

Due to the extensive use of antibiotics, the increase of infections caused by antibiotic-resistant bacteria is now a global health concern. Phages have proven useful for treating bacterial infections and represent a promising alternative or complement to antibiotic treatment. Yet, other alternatives exist, such as bacteria-produced non-replicative protein complexes that can kill their targeted bacteria by puncturing their membrane (Tailocins). To expand the repertoire of Tailocins available, we suggest a new approach that transforms phages into Tailocins. Here, we genetically engineered the virulent Ackermannviridae phage S117, as well as temperate phages Fels-1, -2 and Gifsy-1 and -2, targeting the food pathogen Salmonella, by deleting the portal vertex or major capsid gene using CRISPR-Cas9. We report the production of Tailocin particles from engineered virulent and temperate phages able to kill their native host. Our work represents a steppingstone that taps into the huge diversity of phages and transforms them into versatile puncturing new antimicrobials.

Producing Tailocins from Phages Using Osmotic Shock and Benzalkonium Chloride.

In the light of the worldwide antimicrobial resistance crisis, new substitutes to antibiotics are urgently needed. Tailocins or phage tail-like bacteriocin particles, produced by bacteria for environmental competition, are a potential antimicrobial alternative to antibiotic treatment. Yet, the availability of characterized Tailocins is limited. We explored the possibility to produce new Tailocins from phage particles, using osmotic shock or chemical treatment by the ammonium quaternary compound benzalkonium chloride on Ackermannviridae phage S117 and using Straboviridae phage T4 as control. We report that phage S117 was resistant to such treatment, while successful production of Tailocins by osmotic shock was achieved for phage T4. Finally, chemical treatment with benzalkonium chloride was inefficient on phage S117 but successfully inactivated phage T4 without production of Tailocins. Further studies are needed to implement such treatments of phages for producing Tailocins with killing activity.

A Glimpse at the Anti-Phage Defenses Landscape in the Foodborne Pathogen Salmonella enterica subsp. enterica serovar Typhimurium.

Bacteriophages, which specifically infect and kill bacteria, are currently used as additives to control pathogens such as Salmonella in human food (PhageGuard S®) or animal feed (SalmoFREE®, Bafasal®). Indeed, salmonellosis is among the most important zoonotic foodborne illnesses. The presence of anti-phage defenses protecting bacteria against phage infection could impair phage applications aiming at reducing the burden of foodborne pathogens such as Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) to the food industry. In this study, the landscape of S. Typhimurium anti-phage defenses was bioinformatically investigated in publicly available genomes using the webserver PADLOC. The primary anti-phage systems identified in S. Typhimurium use nucleic acid degradation and abortive infection mechanisms. Reference systems were identified on an integrative and conjugative element, a transposon, a putative integrative and mobilizable element, and prophages. Additionally, the mobile genetic elements (MGEs) containing a subset of anti-phage systems were found in the Salmonella enterica species. Lastly, the MGEs alone were also identified in the Enterobacteriaceae family. The presented diversity assessment of the anti-phage defenses and investigation of their dissemination through MGEs in S. Typhimurium constitute a first step towards the design of preventive measures against the spread of phage resistance that may hinder phage applications.

Genomic and Phenotypic Characterization of Clostridium botulinum Isolates from an Infant Botulism Case Suggests Adaptation Signatures to the Gut.

In early life, the immature human gut microbiota is prone to colonization by pathogens that are usually outcompeted by mature microbiota in the adult gut. Colonization and neurotoxin production by a vegetative Clostridium botulinum culture in the gut of an infant can lead to flaccid paralysis, resulting in a clinical outcome known as infant botulism, a potentially life-threatening condition. Beside host factors, little is known of the ecology, colonization, and adaptation of C. botulinum to the gut environment. In our previous report, an infant with intestinal botulism was shown to be colonized by neurotoxigenic C. botulinum culture for 7 months. In an effort to gain ecological and evolutionary insights into this unusually long gut colonization by C. botulinum, we analyzed and compared the genomes of C. botulinum isolates recovered from the infant feces during the course of intoxication and isolates from the infant household dust. A number of observed mutations and genomic alterations pinpointed at phenotypic traits that may have promoted colonization and adaptation to the gut environment and to the host. These traits include motility, quorum-sensing, sporulation, and carbohydrate metabolism. We provide novel perspectives and suggest a tentative model of the pathogenesis of C. botulinum in infant botulism. IMPORTANCE While the clinical aspects of infant botulism and the mode of action of BoNT have been thoroughly investigated, little is known on the pathogenesis and adaptive mechanisms of C. botulinum in the gut. Here, we provide for the first time a comprehensive view on the genomic dynamics and plasticity of C. botulinum over time in a case of infant botulism. The genomic and phenotypic analysis of C. botulinum isolates collected during the disease course offers an unprecedented view of C. botulinum ecology, evolution, and pathogenesis and may be instrumental in developing novel strategies for prevention and treatment of toxicoinfectious botulism.

Clostridium botulinum type C, D, C/D, and D/C: An update.

Clostridium botulinum is the main causative agent of botulism, a neurological disease encountered in humans as well as animals. Nine types of botulinum neurotoxins (BoNTs) have been described so far. Amongst these "toxinotypes," the A, the B and E are the most frequently encountered in humans while the C, D, C/D and D/C are mostly affecting domestic and wild birds as well as cattle. In France for instance, many cases and outbreaks are reported in these animal species every year. However, underestimation is very likely at least for avifauna species where the detection of dead animals can be challenging. Knowledge about BoNTs C, D, C/D, and D/C and the diseases they cause in animals and humans is still scarce and unclear. Specifically, the potential role of animal botulism outbreaks in cattle and poultry as a source of human illness needs to be further assessed. In this narrative review, we present the current knowledge about toxinotypes C, D, C/D, and D/C in cattle and poultry with, amongst various other aspects, their epidemiological cycles. We also discuss the zoonotic potential of these toxinotypes and some possible ways of risk mitigation. An adapted and effective management of botulism outbreaks in livestock also requires a better understanding of these less common and known toxinotypes.

Exploration of the Diversity of Clustered Regularly Interspaced Short Palindromic Repeats-Cas Systems in Clostridium novyi sensu lato.

Classified as the genospecies Clostridium novyi sensu lato and distributed into four lineages (I-IV), Clostridium botulinum (group III), Clostridium novyi, and Clostridium haemolyticum are clostridial pathogens that cause animal diseases. Clostridium novyi sensu lato contains a large mobilome consisting of plasmids and circular bacteriophages. Here, we explored clustered regularly interspaced short palindromic repeats (CRISPR) arrays and their associated proteins (Cas) to shed light on the link between evolution of CRISPR-Cas systems and the plasmid and phage composition in a study of 58 Clostridium novyi sensu lato genomes. In 55 of these genomes, types I-B (complete or partial), I-D, II-C, III-B, III-D, or V-U CRISPR-Cas systems were detected in chromosomes as well as in mobile genetic elements (MGEs). Type I-B predominated (67.2%) and was the only CRISPR type detected in the Ia, III, and IV genomic lineages. Putative type V-U CRISPR Cas14a genes were detected in two different cases: next to partial type-IB CRISPR loci on the phage encoding the botulinum neurotoxin (BoNT) in lineage Ia and in 12 lineage II genomes, as part of a putative integrative element related to a phage-inducible chromosomal island (PICI). In the putative PICI, Cas14a was associated with CRISPR arrays and restriction modification (RM) systems as part of an accessory locus. This is the first time a PICI containing such locus has been detected in C. botulinum. Mobilome composition and dynamics were also investigated based on the contents of the CRISPR arrays and the study of spacers. A large proportion of identified protospacers (20.2%) originated from Clostridium novyi sensu lato (p1_Cst, p4_BKT015925, p6_Cst, CWou-2020a, p1_BKT015925, and p2_BKT015925), confirming active exchanges within this genospecies and the key importance of specific MGEs in Clostridium novyi sensu lato.

Subtypes of tail spike proteins predicts the host range of Ackermannviridae phages.

Phages belonging to the Ackermannviridae family encode up to four tail spike proteins (TSPs), each recognizing a specific receptor of their bacterial hosts. Here, we determined the TSPs diversity of 99 Ackermannviridae phages by performing a comprehensive in silico analysis. Based on sequence diversity, we assigned all TSPs into distinctive subtypes of TSP1, TSP2, TSP3 and TSP4, and found each TSP subtype to be specifically associated with the genera (Kuttervirus, Agtrevirus, Limestonevirus, Taipeivirus) of the Ackermannviridae family. Further analysis showed that the N-terminal XD1 and XD2 domains in TSP2 and TSP4, hinging the four TSPs together, are preserved. In contrast, the C-terminal receptor binding modules were only conserved within TSP subtypes, except for some Kuttervirus TSP1s and TSP3s that were similar to specific TSP4s. A conserved motif in TSP1, TSP3 and TSP4 of Kuttervirus phages may allow recombination between receptor binding modules, thus altering host recognition. The receptors for numerous uncharacterized phages expressing TSPs in the same subtypes were predicted using previous host range data. To validate our predictions, we experimentally determined the host recognition of three of the four TSPs expressed by kuttervirus S117. We confirmed that S117 TSP1 and TSP2 bind to their predicted host receptors, and identified the receptor for TSP3, which is shared by 51 other Kuttervirus phages. Kuttervirus phages were thus shown encode a vast genetic diversity of potentially exchangeable TSPs influencing host recognition. Overall, our study demonstrates that comprehensive in silico and host range analysis of TSPs can predict host recognition of Ackermannviridae phages.

Closing Clostridium botulinum Group III Genomes Using Long-Read Sequencing.

Clostridium botulinum group III is the anaerobic Gram-positive bacterium producing the deadly neurotoxin responsible for animal botulism. Here, we used long-read sequencing to produce four complete genomes from Clostridium botulinum group III neurotoxin types C, D, C/D, and D/C. The protocol for obtaining high-molecular-weight DNA from C. botulinum group III is described.

Molecular subtyping of European swine influenza viruses and scaling to high-throughput analysis.

BACKGROUND: Swine influenza is a respiratory infection of pigs that may have a significant economic impact in affected herds and pose a threat to the human population since swine influenza A viruses (swIAVs) are zoonotic pathogens. Due to the increasing genetic diversity of swIAVs and because novel reassortants or variants may become enzootic or have zoonotic implications, surveillance is strongly encouraged. Therefore, diagnostic tests and advanced technologies able to identify the circulating strains rapidly are critically important. RESULTS: Several reverse transcription real-time PCR assays (RT-qPCRs) were developed to subtype European swIAVs in clinical samples previously identified as containing IAV genome. The RT-qPCRs aimed to discriminate HA genes of four H1 genetic lineages (H1av, H1hu, H1huΔ146-147, H1pdm) and one H3 lineage, and NA genes of two N1 lineages (N1, N1pdm) and one N2 lineage. After individual validation, each RT-qPCR was adapted to high-throughput analyses in parallel to the amplification of the IAV M gene (target for IAV detection) and the ß-actin gene (as an internal control), in order to test the ten target genes simultaneously on a large number of clinical samples, using low volumes of reagents and RNA extracts. CONCLUSION: The RT-qPCRs dedicated to IAV molecular subtyping enabled the identification of swIAVs from the four viral subtypes that are known to be enzootic in European pigs, i.e. H1avN1, H1huN2, H3N2 and H1N1pdm. They also made it possible to discriminate a new antigenic variant (H1huN2Δ146-147) among H1huN2 viruses, as well as reassortant viruses, such as H1huN1 or H1avN2 for example, and virus mixtures. These PCR techniques exhibited a gain in sensitivity as compared to end-point RT-PCRs, enabling the characterization of biological samples with low genetic loads, with considerable time saving. Adaptation to high-throughput analyses appeared effective, both in terms of specificity and sensitivity. This new development opens novel perspectives in diagnostic capacities that could be very useful for swIAV surveillance and large-scale epidemiological studies.

Investigation of Clostridium botulinum group III's mobilome content.

Clostridium botulinum group III is mainly responsible for botulism in animals. It could lead to high animal mortality rates and, therefore, represents a major environmental and economic concern. Strains of this group harbor the botulinum toxin locus on an unstable bacteriophage. Since the release of the first complete C. botulinum group III genome sequence (strain BKT015925), strains have been found to contain others mobile elements encoding for toxin components. In this study, seven assays targeting toxin genes present on the genetic mobile elements of C. botulinum group III were developed with the objective to better characterize C. botulinum group III strains. The investigation of 110 C. botulinum group III strains and 519 naturally contaminated samples collected during botulism outbreaks in Europe showed alpha-toxin and C2-I/C2-II markers to be systematically associated with type C/D bont-positive samples, which may indicate an important role of these elements in the pathogenicity mechanisms. On the contrary, bont type D/C strains and the related positive samples appeared to contain almost none of the markers tested. Interestingly, 31 bont-negative samples collected on farms after a botulism outbreak revealed to be positive for some of the genetic mobile elements tested. This suggests loss of the bont phage, either in farm environment after the outbreak or during laboratory handling.

Draft Genome Sequences of Five Brazilian Clostridium botulinum Group III Type D/C Strains.

Animal botulism is mainly associated with Clostridium botulinum group III-producing neurotoxin types C, C/D, D, and D/C. In this report, we present the draft genome sequences of the first five strains of Clostridium botulinum type D/C isolated in Brazil and used for vaccination purposes.

Draft Genome Sequences of 12 Feline Bartonella henselae Isolates.

Bartonella henselae is the main causative agent of cat scratch disease. In this report, we present the draft genome sequences of 12 strains of Bartonella henselae originating from the United States, Denmark, and France. These strains were isolated from cats and belonged to either 16S rRNA genotype I or 16S rRNA genotype II.

Development and Validation of a New Reliable Method for the Diagnosis of Avian Botulism.

Liver is a reliable matrix for laboratory confirmation of avian botulism using real-time PCR. Here, we developed, optimized, and validated the analytical steps preceding PCR to maximize the detection of Clostridium botulinum group III in avian liver. These pre-PCR steps included enrichment incubation of the whole liver (maximum 25 g) at 37°C for at least 24 h in an anaerobic chamber and DNA extraction using an enzymatic digestion step followed by a DNA purification step. Conditions of sample storage before analysis appear to have a strong effect on the detection of group III C. botulinum strains and our results recommend storage at temperatures below -18°C. Short-term storage at 5°C is possible for up to 24 h, but a decrease in sensitivity was observed at 48 h of storage at this temperature. Analysis of whole livers (maximum 25 g) is required and pooling samples before enrichment culturing must be avoided. Pooling is however possible before or after DNA extraction under certain conditions. Whole livers should be 10-fold diluted in enrichment medium and homogenized using a Pulsifier® blender (Microgen, Surrey, UK) instead of a conventional paddle blender. Spiked liver samples showed a limit of detection of 5 spores/g liver for types C and D and 250 spores/g for type E. Using the method developed here, the analysis of 268 samples from 73 suspected outbreaks showed 100% specificity and 95.35% sensitivity compared with other PCR-based methods considered as reference. The mosaic type C/D was the most common neurotoxin type found in examined samples, which included both wild and domestic birds.

Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry.

Botulinum neurotoxin (BoNT) serotypes C and D and their mosaic variants CD and DC cause severe cases of botulism in animal husbandry and wildlife. Epidemiological data on the exact serotype or toxin variant causing outbreaks are rarely available, mainly because of their high sequence identity and the lack of fast and specific screening tools to detect and differentiate the four similar toxins. To fill this gap, we developed four highly specific sandwich enzyme-linked immunosorbent assays (ELISAs) able to detect and differentiate botulinum neurotoxins type BoNT/C, D, CD, and DC based on four distinct combinations of specific monoclonal antibodies targeting both conserved and divergent subdomains of the four toxins. Here, highly sensitive detection with detection limits between 2 and 24 pg mL(-1) was achieved. The ELISAs were extensively validated and results were compared with data obtained by quantitative real-time PCR using a panel of Clostridium botulinum strains, real sample materials from veterinary botulism outbreaks, and non-BoNT-producing Clostridia. Additionally, in order to verify the results obtained by ELISA screening, the new monoclonal antibodies were used for BoNT enrichment and subsequent detection (i) on a functional level by endopeptidase mass spectrometry (Endopep-MS) assays and (ii) on a protein sequence level by LC-MS/MS spectrometry. Based on all technical information gathered in the validation study, the four differentiating ELISAs turned out to be highly reliable screening tools for the rapid analysis of veterinary botulism cases and should aid future field investigations of botulism outbreaks and the acquisition of epidemiological data.

New Insights into the Genetic Diversity of Clostridium botulinum Group III through Extensive Genome Exploration.

Animal botulism is caused by group III Clostridium botulinum strains producing type C and D toxins, or their chimeric forms C/D and D/C. Animal botulism is considered an emerging disease in Europe, notably in poultry production. Before our study, 14 genomes from different countries were available in the public database, but none were from France. In order to investigate the genetic relationship of French strains with different geographical areas and find new potential typing targets, 17 strains of C. botulinum group III were sequenced (16 from France and one from New Caledonia). Fourteen were type C/D strains isolated from chickens, ducks, guinea fowl and turkeys and three were type D/C strains isolated from cattle. The New Caledonian strain was a type D/C strain. Whole genome sequence analysis showed the French strains to be closely related to European strains from C. botulinum group III lineages Ia and Ib. The investigation of CRISPR sequences as genetic targets for differentiating strains in group III proved to be irrelevant for type C/D due to a deficient CRISPR/Cas mechanism, but not for type D/C. Conversely, the extrachromosomal elements of type C/D strains could be used to generate a genetic ID card. The highest level of discrimination was achieved with SNP core phylogeny, which allowed differentiation up to strain level and provide the most relevant information for genetic epidemiology studies and discrimination.

Livers provide a reliable matrix for real-time PCR confirmation of avian botulism.

Diagnosis of avian botulism is based on clinical symptoms, which are indicative but not specific. Laboratory investigations are therefore required to confirm clinical suspicions and establish a definitive diagnosis. Real-time PCR methods have recently been developed for the detection of Clostridium botulinum group III producing type C, D, C/D or D/C toxins. However, no study has been conducted to determine which types of matrices should be analyzed for laboratory confirmation using this approach. This study reports on the comparison of different matrices (pooled intestinal contents, livers, spleens and cloacal swabs) for PCR detection of C. botulinum. Between 2013 and 2015, 63 avian botulism suspicions were tested and 37 were confirmed as botulism. Analysis of livers using real-time PCR after enrichment led to the confirmation of 97% of the botulism outbreaks. Using the same method, spleens led to the confirmation of 90% of botulism outbreaks, cloacal swabs of 93% and pooled intestinal contents of 46%. Liver appears to be the most reliable type of matrix for laboratory confirmation using real-time PCR analysis.

Draft Genome Sequences of 17 French Clostridium botulinum Group III Strains.

Animal botulism is mainly associated with Clostridium botulinum group III strains producing neurotoxin types C, C/D, D, and D/C. In this report, we present the draft genome sequences of fourteen strains of Clostridium botulinum producing type C/D and two strains producing type D/C isolated in France, and one strain producing type D/C that originated from New Caledonia.