Angelo Romano

Shotgun Metagenomics and Volatilome Profile of the Microbiota of Fermented Sausages

ABSTRACT Changes in the microbial gene content and abundance can be analyzed to detect shifts in the microbiota composition due to the use of a starter culture in the food fermentation process, with the consequent shift of key metabolic pathways directly connected with product acceptance. Meat fermentation is a complex process involving microbes that metabolize the main components in meat. The breakdown of carbohydrates, proteins, and lipids can lead to the formation of volatile organic compounds (VOCs) that can drastically affect the organoleptic characteristics of the final products. The present meta-analysis, performed with the shotgun DNA metagenomic approach, focuses on studying the microbiota and its gene content in an Italian fermented sausage produced by using a commercial starter culture (a mix of Lactobacillus sakei and Staphylococcus xylosus), with the aim to discover the connections between the microbiota, microbiome, and the release of volatile metabolites during ripening. The inoculated fermentation with the starter culture limited the development of Enterobacteriaceae and reduced the microbial diversity compared to that from spontaneous fermentation. KEGG database genes associated with the reduction of acetaldehyde to ethanol (EC 1.1.1.1), acetyl phosphate to acetate (EC 2.7.2.1), and 2,3-butanediol to acetoin (EC 1.1.1.4) were most abundant in inoculated samples (I) compared to those in spontaneous fermentation samples (S). The volatilome profiles were highly consistent with the abundance of the genes; elevated acetic acid (1,173.85 μg/kg), ethyl acetate (251.58 μg/kg), and acetoin (1,100.19 μg/kg) were observed in the presence of the starters at the end of fermentation. Significant differences were found in the liking of samples based on flavor and odor, suggesting a higher preference by consumers for the spontaneous fermentation samples. Inoculated samples exhibited the lowest scores for the liking data, which were clearly associated with the highest concentration of acetic acid. IMPORTANCE We present an advance in the understanding of meat fermentation by coupling DNA sequencing metagenomics and metabolomics approaches to describe the microbial function during this process. Very few studies using this global approach have been dedicated to food, and none have examined sausage fermentation, underlying the originality of the study. The starter culture drastically affected the organoleptic properties of the products. This finding underlines the importance of starter culture selection that takes into consideration the functional characteristics of the microorganism to optimize production efficiency and product quality.

Food safety and sustainable nutrition workshops: educational experiences for primary school children in Turin, Italy

European control and prevention policies are focused to guarantee a high level of protection of consumers’ health. Food-borne diseases as obesity, diabetes, food allergy, and food-borne outbreaks are increasing. To prevent food-borne diseases, it is fundamental to involve consumers, in particular children, in educational experiences aimed to learn the proper behaviours to be applied. In this context, we designed and performed 5 educational workshops about food safety, hidden allergens in food and nutrition aimed to involve children attending primary and summer school. These experiences let us collect observations about children knowledge and behaviours. From May to October 2015, a total of 1708 children aged 6 to 11 years joined our workshops. Children were involved in listening activities, laboratory experiments, handling games and sensory experiences. All participants were familiar with food allergy and were interested to know how to behave with allergic people. Children showed great curiosity in discovering that many foods normally contain live bacteria. Less than 25% of children reported to skip breakfast, to have it watching TV or to spend few minutes for it. Many of them (>75%) thought that fruits and vegetables are all year-round available and are not related to a specific period. Very few participants (<25%) knew that freezing is the treatment to be applied to make fresh fish safe from parasites. Children involved in food safety and nutrition educational experiences have the opportunity to increase their awareness about the correct behaviours to prevent food-borne diseases and to improve their own critical thinking about food consumption.

Changes in the gut microbiota composition during pregnancy in patients with gestational diabetes mellitus (GDM)

Gestational diabetes mellitus (GDM), a common pregnancy complication, is associated with an increased risk of maternal/perinatal outcomes. We performed a prospective observational explorative study in 41 GDM patients to evaluate their microbiota changes during pregnancy and the associations between the gut microbiota and variations in nutrient intakes, anthropometric and laboratory variables. GDM patients routinely received nutritional recommendations according to guidelines. The fecal microbiota (by 16S amplicon-based sequencing), was assessed at enrolment (24–28 weeks) and at 38 weeks of gestational age. At the study end, the microbiota α-diversity significantly increased (P < 0.001), with increase of Firmicutes and reduction of Bacteroidetes and Actinobacteria. Patients who were adherent to the dietary recommendations showed a better metabolic and inflammatory pattern at the study-end and a significant decrease in Bacteroides. In multiple regression models, Faecalibacterium was significantly associated with fasting glucose; Collinsella (directly) and Blautia (inversely) with insulin, and with Homeostasis-Model Assessment Insulin-Resistance, while Sutterella with C-reactive protein levels. Consistent with this latter association, the predicted metagenomes showed a correlation between those taxa and inferred KEGG genes associated with lipopolysaccharide biosynthesis. A higher bacterial richness and strong correlations between pro-inflammatory taxa and metabolic/inflammatory variables were detected in GDM patients across pregnancy. Collectively these findings suggest that the development of strategies to modulate the gut microbiota might be a potentially useful tool to impact on maternal metabolic health.

A Set of Multiplex Polymerase Chain Reactions for Genomic Detection of Nine Edible Insect Species in Foods

Abstract On 1 January 2018, a new regulation on ‘Novel Food’ has come into application in the EU. Insects and insect-based products are therefore included among the categories of food which constitute novel foods. Insects are nutrient-rich, produce fewer greenhouse gases and ammonia than conventional livestock, and have high feed conversion efficiency. Insects may be an alternative food source in the near future, but consideration of insects as a food requires scrutiny due to the risk of allergens. The aim of the present study was to develop a set of multiplex polymerase chain reaction (PCR) to detect nine edible insect species directly in foods. Four sets of mPCRs were designed to detect Locusta migratoria migratorioides (Reiche & Fairmaire, 1849) (Orthoptera: Acrididae), Tenebrio molitor (Linnaeus, 1758) (Coleoptera: Tenebrionidae) (mPCR-I), Acheta domesticus (Linnaeus, 1758) (Orthoptera: Gryllidae), Bombyx mori (Linnaeus, 1758) (Lepidoptera: Bombycidae (mPCR-II), Alphitobius diaperinus (Panzer, 1797) (Coleoptera: Tenebrionidae), Schistocerca gregaria (Forskål, 1775) (Orthoptera: Acrididae), Zophobas atratus (Fabricius, 1775) (Coleoptera: Tenebrionidae) (mPCR-III), Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae), and Gryllodes sigillatus (Walker, 1869) (Orthoptera: Gryllidae) (mPCR-IV). Results demonstrate that the panel of mPCRs allowed a rapid genetic identification of the insect species and has proved to be a sensible and highly discriminatory method. The assay is a potential tool in issues related to the labeling of products and food safety, in case of allergic consumers.

Case report of a pustular dermatitis outbreak in sheep: Clinical and food safety considerations

The objective of this report is to describe an outbreak of pustular dermatitis in a flock of about 200 sheep, its clinical evolution and food safety implications. The onset of the symptoms was sudden and the lesions spread very quickly from ewe to ewe, so that in about 3 days almost all of the lactating sheep were stricken. Pustules from 5 different animals, six milk samples, two cheese samples, teat cup samples from the milking machine and farmer’s hands were analysed. A pure culture of Staphylococcus aureus, producing staphylococcal enterotoxin (SE) C, was isolated from pustules. Milk and cheese showed a contamination by coagulase positive staphylococci <15 and 30 colony forming units respectively and the absence of SE. Farmer’s hands and teat cups samples resulted negative for coagulase positive staphylococci. Therapy with daily topical medicaments was prescribed and a prophylactic intervention was suggested by the administration of an autovaccine. The low level of milk and cheese contamination and the absence of SE in cheese supported the decision to not advise the farmer to recall cheese produced with milk from affected animals.

Outbreak of febrile gastroenteritis caused by Listeria monocytogenes 1/2a in sliced cold beef ham, Italy, May 2016

In May 2016, two separate clusters of febrile gastroenteritis caused by Listeria monocytogenes were detected by the local health authority in Piedmont, in northern Italy. We carried out epidemiological, microbiological and traceback investigations to identify the source. The people affected were students and staff members from two different schools in two different villages located in the Province of Turin; five of them were hospitalised. The epidemiological investigation identified a cooked beef ham served at the school canteens as the source of the food-borne outbreak. L. monocytogenes was isolated from the food, the stools of the hospitalised pupils and the environment of the factory producing the cooked beef ham. All isolates except one were serotype 1/2a, shared an indistinguishable PFGE pattern and were 100% identical by whole genome sequencing (WGS). By combining a classical epidemiological approach with both molecular subtyping and WGS techniques, we were able to identify and confirm a Listeria gastroenteritis outbreak associated with consumption of sliced cold beef ham.

Build the Read: A Hands-On Activity for Introducing Microbiology Students to Next-Generation DNA Sequencing and Bioinformatics †

Next-Generation Sequencing (NGS) is the current standard for providing genomic data, by virtue of the ability of the technology to generate a considerable amount of information rapidly and at low cost. The data generated can be of key importance to research and addressing issues in public health and, thus, is relevant to society. Unsurprisingly, content relating to the principle and chemistry underlying Next-Generation Sequencing is presented to almost every microbiology-related class, to professionals across multiple fields and, to the general public as popular science. The most commonly utilized NGS platforms (MiSeq, NextSeq and HighSeq) are those provided by Illumina. In this paper, we describe a hands-on activity for students to represent the chemistry underlying Illumina-based NGS, by creating representative reads using LEGO blocks, to link indexes, assemble the sequence and ‘identify’ the bacteria from which the DNA originated, thereby, in the process introducing the participants to the basic principles of bioinformatics.

Whole-Genome Shotgun Sequence of Salmonella bongori, First Isolated in Northwestern Italy

ABSTRACT This study describes the whole-genome shotgun sequence of Salmonella bongori 48:z35:–, originally isolated from a 1-year-old symptomatic patient in northwest Italy, a typically nonendemic area. The draft genome sequence contained 4.56 Mbp and the G+C content was 51.27%.

Draft genome sequences of four Yersinia enterocolitica strains, isolated from wild ungulate carcasses

ABSTRACT This study describes the draft genome sequences of four Yersinia enterocolitica strains, originally isolated from ungulate carcasses. These isolates were typed biochemically and two were determined to be highly virulent (biotype 1B). The draft genome sequences had a mean size of 4.77 Mb and a mean G+C content of 47.1%.