Novel Experimental Methods for the Investigation of Hermetia illucens (Diptera: Stratiomyidae) Larvae.

Large-scale insect rearing for food and feed production can be improved by understanding diet digestion and host-microbe interactions. To examine these processes in black soldier fly (Hermetia illucens L.; Diptera: Stratiomyidae) larvae, two protocols were developed. Protocol 1 describes a method to produce viable, sterile black soldier fly larvae and a gentle method for diet sterilization. Sterile black soldier fly larvae can be used to study the diverse role of microbes in larval development. Nutrient requirements of sterile black soldier fly larvae are met only through diet. Viable sterile black soldier fly larvae were consistently generated using a four-step treatment with alternating immersions of eggs for 2 min each in ethanol (70%) and sodium hypochlorite (0.6%), over two cycles. A nonthermal method of diet sterilization, namely high-energy electron beam (HEEB) treatment, was introduced. Subsequently, growth of sterile black soldier fly larvae was observed on the HEEB-treated diets (40, 60, and 40% of replicates with poultry feed, liver pie, and an artificial diet, respectively) but not on autoclaved diets. In Protocol 2, we propose a novel method to collect frass from individual larvae. We then measured the metabolites in frass, using high-pressure liquid chromatography. Results on metabolites confirmed the influence of digestion. For instance, succinate increased from 1 to 2 and 7 µmol/g sample from diet to gut homogenate and frass, respectively. The collection method is a promising tool to estimate the diet and nutrient requirements of black soldier fly larvae, thus increasing the performance and reliability of black soldier fly larvae rearing. We discuss in detail the possible applications and limitations of our methods in black soldier fly larvae research.

Phylogenetic, epidemiological and functional analyses of the Streptococcus bovis/Streptococcus equinus complex through an overarching MLST scheme.

BACKGROUND: The Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises seven (sub)species classified as human and animal commensals, emerging opportunistic pathogens and food fermentative organisms. Changing taxonomy, shared habitats, natural competence and evidence for horizontal gene transfer pose difficulties for determining their phylogeny, epidemiology and virulence mechanisms. Thus, novel phylogenetic and functional classifications are required. An SBSEC overarching multi locus sequence type (MLST) scheme targeting 10 housekeeping genes was developed, validated and combined with host-related properties of adhesion to extracellular matrix proteins (ECM), activation of the immune responses via NF-KB and survival in simulated gastric juice (SGJ). RESULTS: Commensal and pathogenic SBSEC strains (n = 74) of human, animal and food origin from Europe, Asia, America and Africa were used in the MLST scheme yielding 66 sequence types and 10 clonal complexes differentiated into distinct habitat-associated and mixed lineages. Adhesion to ECMs collagen I and mucin type II was a common characteristic (23 % of strains) followed by adhesion to fibronectin and fibrinogen (19.7 %). High adhesion abilities were found for East African dairy and human blood isolate branches whereas commensal fecal SBSEC displayed low adhesion. NF-KB activation was observed for a limited number of dairy and blood isolates suggesting the potential of some pathogenic strains for reduced immune activation. Strains from dairy MLST clades displayed the highest relative survival to SGJ independently of dairy adaptation markers lacS/lacZ. CONCLUSION: Combining phylogenetic and functional analyses via SBSEC MLST enabled the clear delineation of strain clades to unravel the complexity of this bacterial group. High adhesion values shared between certain dairy and blood strains as well as the behavior of NF-KB activation are concerning for specific lineages. They highlighted the health risk among shared lineages and establish the basis to elucidate (zoonotic-) transmission, host specificity, virulence mechanisms and enhanced risk assessment as pathobionts in an overarching One Health approach.

Co-cultivation is a powerful approach to produce a robust functionally designed synthetic consortium as a live biotherapeutic product (LBP).

The success of fecal microbiota transplants (FMT) has provided the necessary proof-of-concept for microbiome therapeutics. Yet, feces-based therapies have many associated risks and uncertainties, and hence defined microbial consortia that modify the microbiome in a targeted manner have emerged as a promising safer alternative to FMT. The development of such live biotherapeutic products has important challenges, including the selection of appropriate strains and the controlled production of the consortia at scale. Here, we report on an ecology- and biotechnology-based approach to microbial consortium construction that overcomes these issues. We selected nine strains that form a consortium to emulate the central metabolic pathways of carbohydrate fermentation in the healthy human gut microbiota. Continuous co-culturing of the bacteria produces a stable and reproducible consortium whose growth and metabolic activity are distinct from an equivalent mix of individually cultured strains. Further, we showed that our function-based consortium is as effective as FMT in counteracting dysbiosis in a dextran sodium sulfate mouse model of acute colitis, while an equivalent mix of strains failed to match FMT. Finally, we showed robustness and general applicability of our approach by designing and producing additional stable consortia of controlled composition. We propose that combining a bottom-up functional design with continuous co-cultivation is a powerful strategy to produce robust functionally designed synthetic consortia for therapeutic use.

Traditional milk transformation schemes in Côte d'Ivoire and their impact on the prevalence of Streptococcus bovis complex bacteria in dairy products.

The Streptococcus bovis/Streptococcus equinus complex (SBSEC) and possibly Streptococcus infantarius subsp. infantarius (Sii) are associated with human and animal diseases. Sii predominate in spontaneously fermented milk products with unknown public health effects. Sii/SBSEC prevalence data from West Africa in correlation with milk transformation practices are limited. Northern Côte d'Ivoire served as study area due to its importance in milk production and consumption and to link a wider Sudano-Sahelian pastoral zone of cross-border trade. We aimed to describe the cow milk value chain and determine Sii/SBSEC prevalence with a cross-sectional study. Dairy production practices were described as non-compliant with basic hygiene standards. The system is influenced by secular sociocultural practices and environmental conditions affecting product properties. Phenotypic and molecular analyses identified SBSEC in 27/43 (62.8%) fermented and 26/67 (38.8%) unfermented milk samples. Stratified by collection stage, fermented milk at producer and vendor levels featured highest SBSEC prevalence of 71.4% and 63.6%, respectively. Sii with 62.8% and 38.8% as well as Streptococcus gallolyticus subsp. macedonicus with 7.0% and 7.5% were the predominant SBSEC species identified among fermented and unfermented milk samples, respectively. The population structure of Sii/SBSEC isolates seems to reflect evolving novel dairy-adapted, non-adapted and potentially pathogenic lineages. Northern Côte d'Ivoire was confirmed as area with high Sii presence in dairy products. The observed production practices and the high diversity of Sii/SBSEC supports in-depth investigations on Sii ecology niche, product safety and related technology in the dairy value chain potentially affecting large population groups across sub-Saharan Africa.

Risk factors for the carriage of Streptococcus infantarius subspecies infantarius isolated from African fermented dairy products.

Streptococcus infantarius subsp. infantarius (Sii) has been identified as predominant lactic acid bacteria in spontaneously fermented dairy products (FDPs) in sub-Saharan Africa including Côte d'Ivoire. However, Sii belongs to the Streptococcus bovis/Streptococcus equinus complex (SBSEC). Most SBSEC members are assumed to be involved as opportunistic pathogens in serious diseases in both humans and animals. A population-based cross-sectional survey, including 385 participants was conducted in Korhogo, northern Côte d'Ivoire, to identify risk factors for Sii fecal carriage, including consumption of local FDPs. A structured questionnaire was used to gather participant's socio-demographic and economic characteristics, their relation to livestock and dietary habits. In addition, fresh stool and milk samples were collected. The identification of Sii was done using a SBSEC-specific PCR assay targeting 16S rRNA and groEL genes. The overall prevalence of SBSEC and Sii carriage was 23.2% (confidence interval CI 95% = 18.9-27.5) and 12.0% (CI 95% = 8.4-15.5) for stool, respectively. Prevalence of Sii was significantly higher in consumers of artisanal butter compared with non-consumers (57.1% vs 10.1%, odds ratio OR: 11.9, 95% CI: 3.9-36.6), as well as in persons handling livestock (OR = 3.9; 95% CI = 1.6-9.3) and livestock primary products (OR = 5.7; 95% CI = 2.3-14.3). The closer contact with livestock was a risk factor for Sii fecal carriage. Sii strains were isolated from fresh and fermented milk products with a prevalence of 30.4% and 45.4%, respectively. Analysis of Sii population structure through the SBSEC multi locus sequence typing assay revealed a close relationship across human and dairy isolates, possibly linked to a Kenyan human isolate. All these outcomes underline the interest of in-depth investigations on the ecology, potential reservoirs and pathways of contamination by Sii at the human-animal-environment interface in comparison to yet to be collected data from Europe, Asia and the Americas to further elucidate the various roles of Sii.