Valerio Giaccone

Multiple-Locus Sequence Typing and Analysis of Toxin Genes in Bacillus cereus Food-Borne Isolates†

ABSTRACT In the present study we characterized 47 food-borne isolates of Bacillus cereus using multilocus sequence typing (MLST). Newly determined sequences were combined with sequences available in public data banks in order to produce the largest data set possible. Phylogenetic analysis was performed on a total of 296 strains for which MLST sequence information is available, and three main lineages—I, II, and III—within the B. cereus complex were identified. With few exceptions, all food-borne isolates were in group I. The occurrence of horizontal gene transfer (HGT) among various strains was analyzed by several statistical methods, providing evidence of widespread lateral gene transfer within B. cereus. We also investigated the occurrence of toxin-encoding genes, focusing on their evolutionary history within B. cereus. Several patterns were identified, indicating a pivotal role of HGT in the evolution of toxin-encoding genes. Our results indicate that HGT is an important element in shaping the population structure of the B. cereus complex. The results presented here also provide strong evidence of reticulate evolution within the B. cereus complex.

Black soldier fly as dietary protein source for broiler quails: apparent digestibility, excreta microbial load, feed choice, performance, carcass and meat traits

In order to expand with validated scientific data the limited knowledge regarding the potential application of insects as innovative feed ingredients for poultry, the present study tested a partial substitution of soya bean meal and soya bean oil with defatted black soldier fly (Hermetia illucens) larvae meal (H) in the diet for growing broiler quails (Coturnix coturnix japonica) on growth performance, mortality, nutrients apparent digestibility, microbiological composition of excreta, feed choice, carcass and meat traits. With this purpose, a total of 450 10-day-old birds were allocated to 15 cages (30 birds/cage) and received three dietary treatments: a Control diet (C) and two diets (H1 and H2) corresponding to 10% and 15% H inclusion levels, respectively (H substituted 28.4% soya bean oil and 16.1% soya bean meal for H1, and 100% soya bean oil and 24.8% soya bean meal for H2, respectively). At 28 days of age, quails were slaughtered, carcasses were weighed, breast muscles were then excised from 50 quails/treatment, weighed, and ultimate pH (pHu) and L*, a*, b* colour values were measured. Breast muscles were then cooked to assess cooking loss and meat toughness. For the digestibility trial, a total of 15 28-day-old quails were assigned to the three feeding groups. The excreta samples were subjected to chemical and microbiological analysis. The same 15 quails were then simultaneously provided with C and H2 diets for a 10-day feed choice trial. Productive performance, mortality and carcass traits were in line with commercial standards and similar in all experimental groups. With the exception of ether extract digestibility, which was lower in H1 group compared with C and H2 (P=0.0001), apparent digestibility of dry matter, CP, starch and energy did not differ among treatments. Microbial composition of excreta was also comparable among the three groups. Feed choice trial showed that quails did not express a preference toward C or H2 diets. Breast meat weight and yield did not differ among C, H1 and H2 quails. Differently, the inclusion of H meal reduced meat pHu compared with C. In conclusion, this study demonstrated that H. illucens larvae meal can partially replace conventional soya bean meal and soya bean oil in the diet for growing broiler quails, thus confirming to be a promising insect protein source for the feed industry. Further research to assess the impact of H meal on intestinal morphology as well as on meat quality and sensory profile would be of utmost importance.

Dietary Spirulina (Arthrospira platensis) and Thyme (Thymus vulgaris) supplementation to growing rabbits: Effects on raw and cooked meat quality, nutrient true retention and oxidative stability

The study evaluated the effect of Spirulina and Thyme dietary supplementation on rabbit meat quality, nutrient true retention and protection against oxidative stress. Rabbits in the control group (C-C) received a non-supplemented pellet throughout the experiment (5-11weeks of age). In the other groups, the pellet contained 5% Spirulina (S), 3% Thyme (T), or both (ST) for either the entire (groups S-S, T-T, ST-ST) or only the final part of the growing period (8-11weeks: groups C-S, C-T, C-ST). Spirulina supplementation increased the γ-linolenic acid content of rabbit meat, whereas Thyme improved the oxidative stability of raw and freeze-dried meat.

Resistance to biocides in Listeria monocytogenes collected in meat-processing environments

The emergence of microorganisms exerting resistance to biocides is a challenge to meat-processing environments. Bacteria can be intrinsically resistant to biocides but resistance can also be acquired by adaptation to their sub-lethal concentrations. Moreover, the presence of biocide resistance determinants, which is closely linked to antibiotic resistance determinants, could lead to co-selection during disinfection practices along the food chain, and select cross-resistant foodborne pathogens. The purpose of this work was to test the resistance of wild strains of Listeria monocytogenes, isolated from pork meat processing plants, toward benzalkonium chloride (BC), used as proxy of quaternary ammonium compounds. Furthermore, the expression of two non-specific efflux pumps genes (lde and mdrL) under biocide exposure was evaluated. L. monocytogenes were isolated from five processing plants located in the Veneto region (northeast of Italy) before and after cleaning and disinfection (C&D) procedures. A total of 45 strains were collected: 36 strains before and nine after the C&D procedures. Collected strains were typed according to MLST and ERIC profiles. Strains sampled in the same site, isolated before, and after the C&D procedures and displaying the same MLST and ERIC profiles were tested for their sensitivity to different concentrations of BC, in a time course assay. The expression of non-specific efflux pumps was evaluated at each time point by qPCR using tufA gene as housekeeping. A differential expression of the two investigated genes was observed: lde was found to be more expressed by the strains isolated before C&D procedures while its expression was dose-dependent in the case of the post C&D procedures strain. On the contrary, the expression of mdrL was inhibited under low biocidal stress (10 ppm BC) and enhanced in the presence of high stress (100 ppm BC). These findings suggests a possible role for C&D procedures to select L. monocytogenes persisters, pointing out the importance of dealing with the identification of risk factors in food plants sanification procedures that might select more tolerant strains.

Students’ Consumption of Beverages and Snacks at School and Away from School: A Case Study in the North East of Italy

In North-East Italy (the Veneto region), several public school nutrition policies have been developed to reduce the consumption of high-caloric snacks and beverages. However, little is known about whether the policies actually influence students’ dietary behaviors. In order to address this point, a multi-center cross-sectional survey of 691 Italian students was conducted. Students completed the Beverage and Snack Questionnaire, which assesses the consumption of beverages and snacks at school and out of school. Three-level Poisson Models with random intercept with students (level 1 units) nested into classroom (level 2 units), and nested into schools (level 3 units), were used to examine the influence of the school setting vs. the out of school environment (independent variable) on students’ consumption of sweet beverages, snacks, milk-based beverages, low-carbohydrate drinks, fruit, and vegetables (dependent variable) (p ≤ 0.05). The results showed a significantly higher consumption of sweet beverages, snacks, milk-based beverages, low-carbohydrate drinks, fruit, and vegetables out-of-the school, suggesting a school-protective association Thus, the policies aimed to limit or deny access to unhealthy foods in the school environment may play an important role in promoting more healthful dietary patterns for school children. Additional studies should be conducted to compare students’ dietary behaviors between schools with nutrition policies to those without nutrition policies.

Black soldier fly as dietary protein source for broiler quails: meat proximate composition, fatty acid and amino acid profile, oxidative status and sensory traits

In the perspective of improving the sustainability of meat production, insects have been rapidly emerging as innovative feed ingredient for some livestock species, including poultry. However, at present, there is still limited knowledge regarding the quality and sensory traits of the derived meat. Therefore, the present study tested the effect of a partial substitution of soya bean meal and oil with defatted black soldier fly (Hermetia illucens) larvae meal (H) in the diet for growing broiler quails (Coturnix coturnix japonica) on meat proximate composition, cholesterol, amino acid and mineral contents, fatty acid profile, oxidative status and sensory characteristics. To this purpose, three dietary treatments were designed: a control diet (C) and two diets (H1 and H2) corresponding to 10% and 15% H inclusion levels, respectively, were fed to growing quails from 10 to 28 days of age. At 28 days of age, quails were slaughtered and breast meat was used for meat quality evaluations. Meat proximate composition, cholesterol content and oxidative status remained unaffected by H supplementation as well as its sensory characteristics and off-flavours perception. Differently, with increasing the dietary H inclusion, the total saturated fatty acid and total monounsaturated fatty acid proportions raised to the detriment of the polyunsaturated fatty acid fraction thus lowering the healthiness of the breast meat. The H2 diet increased the contents of aspartic acid, glutamic acid, alanine, serine, tyrosine and threonine thus further enhancing the biological value of the meat protein. As a direct result of the dietary content of Ca and P, the meat of quails fed with the highest H level, displayed the highest Ca and the lowest P values. Therefore, meat quality evaluations confirmed H to be a promising insect protein source for quails. The only potential drawback from feeding H to broiler quails regarded the fatty acid profile of the meat, therefore requiring further research efforts to understand to what extent the fatty acid profile of H can be improved.

Effect of Digestarom® Dietary Supplementation on the Reproductive Performances of Rabbit Does: Preliminary Results

The study investigated the effect of Digestarom® dietary supplementation on the reproductive performances of rabbit does. Pannon Ka (maternal line) multiparous does were randomly divided into two dietary groups since insemination and fed ad libitum. In the first group (n=51), rabbit does were fed with a commercial diet (C), whereas in the other group (n=52) they received the same diet supplemented with 300 mg/kg of Digestarom® (D). The experiment lasted for two reproductive cycles (kindling 1=K1; kindling 2=K2). Body weight of does and litter size (kits born total, alive, stillborn) were recorded at kindling. Litter size and litter weight were registered at 7, 14, 21 days of age after nursing, and the average individual weight of kits was calculated. Kits’ mortality was recorded daily. At K1, rabbit does performances were unaffected by dietary treatments. During K2, D does were significantly lighter than C ones (P<0.05) and displayed a lower kindling rate (P<0.05). Digestarom® did not improve the reproductive performance of rabbit does. They seemed to dislike the D diet in K1 and such behavior could have led to the negative results in K2. Further studies should focus on feed acceptance, dose-dependent effect, physiological adaptation and in vivo oxidative status of does. Finally, several consecutive reproductive cycles are recommended to test the efficacy of new feed supplements.

Microbiological Quantitative Risk Assessment and Food Safety: An Update

Quantitative Risk Assessment is one of three components of Risk Analysis (RA); the others being Risk Management and Risk Communication. Initially, in the 1970s, risk analysis was mainly applied to financial/technological risks. Later on, it was used to address chemical hazards in food products. Since the middle of the 1990s RA has gained international acceptance as the most effective tool for managing microbiological hazards in food and has emerged as a structured model for improving food control systems with the objectives of producing safer food, and for facing the increasing incidence of foodborne illnesses, as well as facilitating domestic and international trade in food. The first official document on risk analysis was developed by FAO/WHO (1995), followed by a series of formal documents on risk management and risk communication (Codex Alimentarius Commission, 1996; FAO/WHO, 1997, 1999). The three components of risk analysis (risk assessment, risk management and risk communication) are not fully separated, but partly overlapping, due to the continuous exchange of information and data among all subjects who participate in the process. Risk assessment can be defined as a structured science-based process to estimate the likelihood and severity of risk with attendant uncertainty. Many organizations recognize four major elements of risk assessment that constitute a logical and sequential pathway: hazard identification, hazard characterization, exposure assessment and hazard characterization. Hazard identification and hazard characterization represent very important phases, but in this short manuscript, we mainly focus on exposure assessment and risk characterization. Lammerding and Fazil (2000) define exposure assessment as “the probability of exposure of a individual or population to a microbiological hazard and the likely ingestion of a dose of the micro-organism.” Exposure assessment (frequency and ingested dose of the microorganism) provides input for the selected dose-response models. Risk characterization, following the definition of Buchanan et al. (2000), is the overall evaluation of the likelihood that a certain population will suffer adverse effects as a result of the hazard and is the integration of the exposure and dose-response assessment. With risk characterization, which represents the final stage of microbial food safety risk assessment, we try to estimate both the probability (risk estimate) and the severity of adverse effects resulting from the ingestion of contaminated food. This probability is dependent on the

Effect of pre- and post-weaning dietary supplementation with Digestarom® herbal formulation on rabbit carcass traits and meat quality

This study evaluated effects of Digestarom® (D) dietary inclusion before weaning (0-5weeks old; BW) and/or after weaning (5-12weeks old; AW) on growing rabbit carcass traits and meat quality. During BW, Pannon-Ka rabbits (does, kits) received two diets: a control diet (C) and one supplemented with 300mg Digestarom®/kg (D). At weaning, each group was divided into 3 dietary sub-groups: CC and DD received C and D diets from 5 to 12weeks of age, whereas DC was fed D from 5 to 8weeks and C from 8 to 12weeks of age (54 rabbits/group; AW). Rabbits were slaughtered at 12weeks of age. Digestarom® supplementation improved carcass yield and body mid part proportion only when administered BW. Rabbits fed D BW had higher hind leg meat cooking losses. Loin meat spiciness and rancidity increased with D both BW and AW. In conclusion, Digestarom(®) herbal formulation was ineffective in improving growing rabbit carcass traits or meat quality.

Approaches to game hygiene in the province Belluno (Italy): from training to meat microbiology

In the last decades, the number of animals shot in the Italian Alps during the regular hunting seasons has dramatically increased, which is particularly the case for wild ungulates. In these areas game is considered an important source of meat, and an increasing interest of consumers for game meat with high added value and favourable nutritional properties is noticeable (Hoffman and Wiklund, 2006). Nevertheless, targeted measures to assure game meat hygiene are still lacking in Italy, and much work is needed to convince hunters and consumers in general of the importance of this topic. This contribution summarises various complementary approaches to game meat hygiene, as initiated in the territory of the province Belluno, in the Italian Eastern Alps, i.e. the centre of the Dolomites, where red deer, roe deer, chamois, mouflon and wild boar are hunted.