Exploitation of Tenebrio molitor larvae as biological factories for human probiotics, an exploratory study

The exploitation of yellow mealworm (Tenebrio molitor) larvae for the bioaugmentation of probiotic Bacillus clausii strains was evaluated during a 7-day rearing period. qPCR was applied to evaluate the persistence and growth of B. clausii in the rearing substrate and larvae (washed and non-washed). Moreover, the effect of freeze-drying of larvae on B. clausii viability was evaluated. The results demonstrated the suitability of yellow mealworm as biological factories for the multiplication of B. clausii through a simple and inexpensive procedure, in view of the further application of larvae as foods and food ingredients. In more detail, an increase in the load of B. clausii was observed during the 7-day rearing of larvae fed wheat middlings spiked with 1 Log cells g−1. Further research is needed to evaluate the most suitable technologies and processing parameters for obtaining yellow mealworm-based ingredients with a stable and active population of probiotic B. clausii

Microbial dynamics in rearing trials of Hermetia illucens larvae fed coffee silverskin and microalgae

In the present study, Hermetia illucens larvae were reared on a main rearing substrate composed of a coffee roasting byproduct (coffee silverskin, Cs) enriched with microalgae (Schizochytrium limacinum or Isochrysis galbana) at various substitution levels. The microbial diversity of the rearing substrates, larvae, and frass (excrement from the larvae mixed with the substrate residue) were studied by the combination of microbial culturing on various growth media and metataxonomic analysis (Illumina sequencing). High counts of total mesophilic aerobes, bacterial spores, presumptive lactic acid bacteria, coagulase-positive cocci, and eumycetes were detected. Enterobacteriaceae counts were low in the rearing diets, whereas higher counts of this microbial family were observed in the larvae and frass. The microbiota of the rearing substrates was characterized by the presence of lactic acid bacteria, including the genera Lactobacillus, Leuconostoc and Weissella. The microbiota of the H. illucens larvae fed Cs was characterized by the dominance of Paenibacillus. H. illucens fed diets containing I. galbana were characterized by the presence of Enterococcus, Lysinibacillus, Morganella, and Paenibacillus, depending on the algae inclusion level, while H. illucens fed diets containing S. limacinum were characterized by high relative abundances of Brevundimonas, Enterococcus, Paracoccus, and Paenibacillus, depending on the algae inclusion level. Brevundimonas and Alcaligenes dominated in the frass from larvae fed I. galbana; the predominance of Brevundimonas was also observed in the frass from larvae fed Schyzochitrium-enriched diets. Based on the results of the present study, an effect of algae nutrient bioactive substances (e.g. polysaccharides, high-unsaturated fatty acids, taurine, carotenoids) on the relative abundance of some of the bacterial taxa detected in larvae may be hypothesized, thus opening new intriguing perspectives for the control of the entomopathogenic species and foodborne human pathogens potentially occurring in edible insects. Further studies are needed to support this hypothesis. Finally, new information on the microbial diversity occurring in insect frass was also obtained

The Hyperphagia Questionnaire: Insights From a Multicentric Validation Study in Individuals With Prader Willi Syndrome

The present study aimed to validate the Italian version of the Hyperphagia Questionnaire (HQ), a 11-items questionnaire developed to assess hyperphagia in individuals with Prader-Willi syndrome (PWS). This is a complex neurodevelopmental disorder characterized by endocrine dysfunction, hypotonia, intellectual disability, psychiatric disorders and obesity

Clinical and molecular characterization of patients affected by Beckwith-Wiedemann spectrum conceived through assisted reproduction techniques

The prevalence of Beckwith-Wiedemann spectrum (BWSp) is tenfold increased in children conceived through assisted reproductive techniques (ART). More than 90% of ART-BWSp patients reported so far display imprinting center 2 loss-of-methylations (IC2-LoM), versus 50% of naturally conceived BWSp patients. We describe a cohort of 74 ART-BWSp patients comparing their features with a cohort of naturally conceived BWSp patients, with the ART-BWSp patients previously described in literature, and with the general population of children born from ART. We found that the distribution of UPD(11)pat was not significantly different in ART and naturally conceived patients. We observed 68.9% of IC2-LoM and 16.2% of mosaic UPD(11)pat in our ART cohort, that strongly differ from the figure reported in other cohorts so far. Since UPD(11)pat likely results from post-fertilization recombination events, our findings allows to hypothesize that more complex molecular mechanisms, besides methylation disturbances, may underlie BWSp increased risk in ART pregnancies. Moreover, comparing the clinical features of ART and non-ART BWSp patients, we found that ART-BWSp patients might have a milder phenotype. Finally, our data show a progressive increase in the prevalence of BWSp over time, paralleling that of ART usage in the last decades

Distribution of Transferable Antibiotic Resistance Genes in Laboratory-Reared Edible Mealworms (Tenebrio molitor L.)

In the present study, the distribution of antibiotic resistance genes in laboratory-reared fresh mealworm larvae (Tenebrio molitor L.), their feeding substrates (carrots and wheatmeal), and frass was assessed. Microbial counts on selective media added with antibiotics highlighted the presence of lactic acid bacteria resistant to ampicillin and vancomycin and, more specifically, enterococci resistant to the latter antibiotic. Moreover, staphylococci resistant to gentamicin, erythromycin, tetracycline, and vancomycin were detected. Enterobacteriaceae resistant to ampicillin and gentamicin were also found, together with Pseudomonadaceae resistant to gentamicin. Some of the genes coding for resistance to macrolide-lincosamide-streptogramin B (MLSB) [erm(A), erm(C)], vancomycin [vanA, vanB], tetracycline [tet(O)], and β-lactams [mecA and blaZ] were absent in all of the samples. For the feeding substrates, organic wheatmeal was positive for tet(S) and tet(K), whereas no AR genes were detected in organic carrots. The genes tet(M), tet(K), and tet(S) were detected in both mealworms and frass, whereas gene aac-aph, coding for resistance to amynoglicosides was exclusively detected in frass. No residues for any of the 64 antibiotics belonging to 10 different drug classes were found in either the organic wheatmeal or carrots. Based on the overall results, the contribution of feed to the occurrence of antibiotic resistance (AR) genes and/or antibiotic-resistant microorganisms in mealworm larvae was hypothesized together with vertical transmission via insect egg smearing

The Italian registry for patients with Prader-Willi syndrome

Background: Prader-Willi syndrome (PWS) is a rare and complex genetic disease, with numerous implications on metabolic, endocrine, neuropsychomotor systems, and with behavioural and intellectual disorders. Rare disease patient registries are important scientific tools (1) to collect clinical and epidemiologic data, (2) to assess the clinical management including the diagnostic delay, (3) to improve patients' care and (4) to foster research to identify new therapeutic solutions. The European Union has recommended the implementation and use of registries and databases. The main aims of this paper are to describe the process of setting up the Italian PWS register, and to illustrate our preliminary results. Materials and methods: The Italian PWS registry was established in 2019 with the aims (1) to describe the natural history of the disease, (2) to determine clinical effectiveness of health care services, (3) to measure and monitor quality of care of patients. Information from six different variables are included and collected into this registry: demographics, diagnosis and genetics, patient status, therapy, quality of life and mortality. Results: A total of 165 patients (50.3% female vs 49.7% male) were included into Italian PWS registry in 2019-2020 period. Average age at genetic diagnosis was 4.6 years; 45.4% of patients was less than 17 years old aged, while the 54.6% was in adult age (> 18 years old). Sixty-one percent of subjects had interstitial deletion of the proximal long arm of paternal chromosome 15, while 36.4% had uniparental maternal disomy for chromosome 15. Three patients presented an imprinting centre defect and one had a de novo translocation involving chromosome 15. A positive methylation test was demonstrated in the remaining 11 individuals but the underlying genetic defect was not identified. Compulsive food-seeking and hyperphagia was present in 63.6% of patients (prevalently in adults); 54.5% of patients developed morbid obesity. Altered glucose metabolism was present in 33.3% of patients. Central hypothyroidism was reported in 20% of patients; 94.7% of children and adolescents and 13.3% of adult patients is undergoing GH treatment. Conclusions: The analyses of these six variables allowed to highlight important clinical aspects and natural history of PWS useful to inform future actions to be taken by national health care services and health professionals

Molecular Mechanisms Generating and Stabilizing Terminal 22q13 Deletions in 44 Subjects with Phelan/McDermid Syndrome

In this study, we used deletions at 22q13, which represent a substantial source of human pathology (Phelan/McDermid syndrome), as a model for investigating the molecular mechanisms of terminal deletions that are currently poorly understood. We characterized at the molecular level the genomic rearrangement in 44 unrelated patients with 22q13 monosomy resulting from simple terminal deletions (72%), ring chromosomes (14%), and unbalanced translocations (7%). We also discovered interstitial deletions between 17–74 kb in 9% of the patients. Haploinsufficiency of the SHANK3 gene, confirmed in all rearrangements, is very likely the cause of the major neurological features associated with PMS. SHANK3 mutations can also result in language and/or social interaction disabilities. We determined the breakpoint junctions in 29 cases, providing a realistic snapshot of the variety of mechanisms driving non-recurrent deletion and repair at chromosome ends. De novo telomere synthesis and telomere capture are used to repair terminal deletions; non-homologous end-joining or microhomology-mediated break-induced replication is probably involved in ring 22 formation and translocations; non-homologous end-joining and fork stalling and template switching prevail in cases with interstitial 22q13.3. For the first time, we also demonstrated that distinct stabilizing events of the same terminal deletion can occur in different early embryonic cells, proving that terminal deletions can be repaired by multistep healing events and supporting the recent hypothesis that rare pathogenic germline rearrangements may have mitotic origin. Finally, the progressive clinical deterioration observed throughout the longitudinal medical history of three subjects over forty years supports the hypothesis of a role for SHANK3 haploinsufficiency in neurological deterioration, in addition to its involvement in the neurobehavioral phenotype of PMS

Time to Switch to Second-line Antiretroviral Therapy in Children With Human Immunodeficiency Virus in Europe and Thailand.

Background: Data on durability of first-line antiretroviral therapy (ART) in children with human immunodeficiency virus (HIV) are limited. We assessed time to switch to second-line therapy in 16 European countries and Thailand. Methods: Children aged <18 years initiating combination ART (≥2 nucleoside reverse transcriptase inhibitors [NRTIs] plus nonnucleoside reverse transcriptase inhibitor [NNRTI] or boosted protease inhibitor [PI]) were included. Switch to second-line was defined as (i) change across drug class (PI to NNRTI or vice versa) or within PI class plus change of ≥1 NRTI; (ii) change from single to dual PI; or (iii) addition of a new drug class. Cumulative incidence of switch was calculated with death and loss to follow-up as competing risks. Results: Of 3668 children included, median age at ART initiation was 6.1 (interquartile range (IQR), 1.7-10.5) years. Initial regimens were 32% PI based, 34% nevirapine (NVP) based, and 33% efavirenz based. Median duration of follow-up was 5.4 (IQR, 2.9-8.3) years. Cumulative incidence of switch at 5 years was 21% (95% confidence interval, 20%-23%), with significant regional variations. Median time to switch was 30 (IQR, 16-58) months; two-thirds of switches were related to treatment failure. In multivariable analysis, older age, severe immunosuppression and higher viral load (VL) at ART start, and NVP-based initial regimens were associated with increased risk of switch. Conclusions: One in 5 children switched to a second-line regimen by 5 years of ART, with two-thirds failure related. Advanced HIV, older age, and NVP-based regimens were associated with increased risk of switch

Exploitation of a polyphasic PCR-DGGE approach forthe investigation of the microbiota of traditional Italian raw milk cheeses.

INTRODUCTION Since the last decade there has been an increasing demand for artisan cheeses manufactured with raw milk and traditional procedures. In raw milk cheeses, fermentation and ripening are carried out by a heterogeneous microbial community, which includes starter lactic acid bacteria (LAB), able to rapidly convert lactose into lactic acid, and non starter LAB (NSLAB) mainly contributing to the development of cheese flavour and aroma. Materials & methods Four traditional cheeses manufactured with local raw milk (Marche region, central Italy) were analysed for physico-chemical (pH, aw), compositional (NaCl, raw protein, fat content) and microbiological traits (occurrence of pathogens and their toxins, and viable counts of lactobacilli, lactococci and thermophilic streptococci). The composition of the LAB population was investigated with a poorly exploited polyphasic PCR-DGGE approach relying on the analysis of the bacterial DNA extracted either from the cheeses or the bulk cells harvested from all the dilution plates used for LAB viable counting. Results & discussion The results of traditional microbiological analyses fully complied with the European legislation [Regulation EC No. 2073/2005]. A high cheese diversity was revealed by physico-chemical, microbiological and PCR-DGGE analyses. As the fingerprints obtained with the two approaches were compared, the analysis of the cultivable communities allowed a significantly higher diversity than the direct approach to be disclosed, although in some cases, species not detected in the bulk cells were identified by analysing the DNA extracted directly from the cheeses. CONCLUSIONS The overall PCR-DGGE results clearly demonstrated that although either approach, taken singly, gives an incomplete picture of the real microbial diversity by introducing bias, their combination allows a greater pool of information to be collected

PCR-DGGE for the profiling of cheese bacterial communities: strengths and weaknesses of a poorly explored combined approach

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) represents a still valid molecular tool for the profiling of complex microbial ecosystems, including cheeses. In the present study, a double PCR-DGGE approach has been applied to the investigation of the bacterial diversity of seven cheese models to objectively assess strengths and weaknesses of such an approach. To that end, the bacterial DNA was extracted directly from both the cheese replicates and the bulks of colonies harvested from the serial dilution agar plates of selective solid media used for the enumeration of presumptive lactobacilli, lactococci and thermophilic cocci, respectively. The results overall collected allowed the main bacterial taxa to be identified and roughly quantified. Rough quantification of the main cultivable species represents a strength of the PCR-DGGE approach applied, whereas its main weaknesses were represented by the low degree of selectivity of the conventional growth media used for cultivation of lactic acid bacteria and the underestimation of the effective microbial diversity occurring in the seven cheese models