Luca Fasolato

Determination of Microbial Diversity of Aeromonas Strains on the Basis of Multilocus Sequence Typing, Phenotype, and Presence of Putative Virulence Genes

ABSTRACT The genus Aeromonas has been described as comprising several species associated with the aquatic environment, which represents their principal reservoir. Aeromonas spp. are commonly isolated from diseased and healthy fish, but the involvement of such bacteria in human infection and gastroenteritis has frequently been reported. The primary challenge in establishing an unequivocal link between the Aeromonas genus and pathogenesis in humans is the extremely complicated taxonomy. With the aim of clarifying taxonomic relationships among the strains and phenotypes, a multilocus sequencing approach was developed and applied to characterize 23 type and reference strains of Aeromonas spp. and a collection of 77 field strains isolated from fish, crustaceans, and mollusks. All strains were also screened for putative determinants of virulence by PCR (ast, ahh1, act, asa1, eno, ascV, and aexT) and the production of acylated homoserine lactones (AHLs). In addition, the phenotypic fingerprinting obtained from 29 biochemical tests was submitted to the nonparametric combination (NPC) test methodology to define the statistical differences among the identified genetic clusters. Multilocus sequence typing (MLST) achieved precise strain genotyping, and the phylogenetic analysis of concatenated sequences delineated the relationship among the taxa belonging to the genus Aeromonas, providing a powerful tool for outbreak traceability, host range diffusion, and ecological studies. The NPC test showed the feasibility of phenotypic differentiation among the majority of the MLST clusters by using a selection of tests or the entire biochemical fingerprinting. A Web-based MLST sequence database (http://pubmlst.org/aeromonas) specific for the Aeromonas genus was developed and implemented with all the results.

Use of Near-Infrared Spectroscopy for Fast Fraud Detection in Seafood: Application to the Authentication of Wild European Sea Bass (Dicentrarchus labrax)

The possibility of using near-infrared spectroscopy (NIRS) for the authentication of wild European sea bass ( Dicentrarchus labrax ) was investigated in this study. Three different chemometric techniques to process the NIR spectra were developed, and their ability to discriminate between wild and farmed sea bass samples was evaluated. One approach used spectral information to directly build the discrimination model in a latent variable space; the second approach first used wavelets to transform the spectral information and subsequently derived the discrimination model using the transformed spectra; in the third approach a cascaded arrangement was proposed whereby very limited chemical information was first estimated from spectra using a regression model, and this estimated information was then used to build the discrimination model in a latent variable space. All techniques showed that NIRS can be used to reliably discriminate between wild and farmed sea bass, achieving the same classification performance as classification methods that use chemical properties and morphometric traits. However, compared to methods based on chemical analysis, NIRS-based classification methods do not require reagents and are simpler, faster, more economical, and environmentally safer. All proposed techniques indicated that the most predictive spectral regions were those related to the absorbance of groups CH, CH(2), CH(3), and H(2)O, which are related to fat, fatty acids, and water content.

Comparison of Visible and Near-Infrared Reflectance Spectroscopy to Authenticate Fresh and Frozen-Thawed Swordfish (Xiphias gladius L)

This study evaluated near-infrared (NIR) and visible-NIR (Vis-NIR) spectroscopy as a way to distinguish fresh (F) from frozen-thawed (T) swordfish cutlets (Xiphias gladius). A total of 90 F and 60 T samples were used. The T samples were stored at a high and low frozen temperature (HT: −10°C; LT: −18°C). Spectra were collected using a Vis-NIR portable spectrophotometer (380–1080 nm) and a NIR monochromator (1100–2500 nm). The percentage of correctly classified samples obtained with Vis-NIR spectroscopy was ≥ 96.7%, whereas that for NIR was ≥ 90.0%. The best classification was observed comparing F and HT samples using Vis-NIR (100 vs. 96.7%, respectively). The more descriptive principal component scores (PCS) of NIR and Vis-NIR were used with a multivariate binary logistic regression. The model with the PCS of the first two Vis-NIR principal components accounted for 81.1% of the classification. Vis-NIR could be a strategic tool to screen the cold treatment of swordfish.

A genomic and transcriptomic approach to investigate the blue pigment phenotype in Pseudomonas fluorescens

Pseudomonas fluorescens is a well-known food spoiler, able to cause serious economic losses in the food industry due to its ability to produce many extracellular, and often thermostable, compounds. The most outstanding spoilage events involving P. fluorescens were blue discoloration of several food stuffs, mainly dairy products. The bacteria involved in such high-profile cases have been identified as belonging to a clearly distinct phylogenetic cluster of the P. fluorescens group. Although the blue pigment has recently been investigated in several studies, the biosynthetic pathway leading to the pigment formation, as well as its chemical nature, remain challenging and unsolved points. In the present paper, genomic and transcriptomic data of 4 P. fluorescens strains (2 blue-pigmenting strains and 2 non-pigmenting strains) were analyzed to evaluate the presence and the expression of blue strain-specific genes. In particular, the pangenome analysis showed the presence in the blue-pigmenting strains of two copies of genes involved in the tryptophan biosynthesis pathway (including trpABCDF). The global expression profiling of blue-pigmenting strains versus non-pigmenting strains showed a general up-regulation of genes involved in iron uptake and a down-regulation of genes involved in primary metabolism. Chromogenic reaction of the blue-pigmenting bacterial cells with Kovacs reagent indicated an indole-derivative as the precursor of the blue pigment. Finally, solubility tests and MALDI-TOF mass spectrometry analysis of the isolated pigment suggested that its molecular structure is very probably a hydrophobic indigo analog.

Polyphenols from olive mill waste affect biofilm formation and motility in Escherichia coli K-12

Olive mill wastes are sources of phenolic compounds with a wide array of biological activities, including antimicrobial effects. A potential option for bioremediation to overcome ecological problems is the reutilization of these natural compounds in food production. The aim of this work was to gain a better understanding of the antimicrobial mode of action of a phenols extract from olive vegetation water (PEOVW) at molecular level by studying Escherichia coli as a model microorganism. Genome‐wide transcriptional analysis was performed on E. coli K‐12 exposed to PEOVW. The repression of genes for flagellar synthesis and the involvement of genes linked to biofilm formation and stress response were observed. Sub‐inhibitory concentrations of PEOVW significantly decreased biofilm formation, swarming and swimming motility, thus confirming the gene expression data. This study provides interesting insights on the molecular action of PEOVW on E. coli K‐12. Given these anti‐biofilm properties and considering that biofilm formation is a serious problem for the food industry and human health, PEOVW has proved to be a high‐value natural product.

Edible insects and young adults in a north-east Italian city an exploratory study

Purpose – The purpose of this paper is to explore the readiness of young Italian people to consume insects and the psychosocial determinants associated with edible insect consumption, in order to evaluate whether insect-based food could be an alternative food source. Design/methodology/approach – An explorative study was conducted, using focus group interviews as the technique for data collection. In total, 32 consumers, aged 20-35 years took part in five focus group interviews. Findings – Significant determinants comprised appearance, farming and sustainability. Participants’ willingness to eat insect-based food is dependent on the form in which the products are presented to them. Crustaceans were frequently mentioned as a comparison in terms of distaste. Lack of practice in preparation was a major barrier to consumption. To expand consumption participants suggested an active role for public health institutions. Even though with some uncertainty, there are some people prepared to consume insects. Researc...

Aeromonas spp.: ubiquitous or specialized bugs?

The genus Aeromonas comprises ubiquitous bacteria that are known to play several roles in the environment. These bacteria were first described as fish pathogens, but their presence was documented in other reservoirs, such as animals and humans. Today, these bacteria are described as emerging pathogens, but their effective role in human pathogenicity is still controversial. In addition, their taxonomy is heavily debated, as species distinction is often difficult to achieve. To study the interspecies relationships and to investigate their connection with the environment, a multilocus sequence typing scheme previously developed for Aeromonas spp. was applied to 258 strains, and the genetic data were analysed by population software. Sampling was a fundamental step, including several of the main sources of Aeromonas: fish, food products and human cases of disease. The objective was to characterize the isolates and to find potential associations among them according to the following: species, sharing of virulence factors, source and adaptation to a specific habitat. The strains were characterized and demonstrated exceptionally high nucleotide variability in the Aeromonas genus. Among the sampled sources, different species distributions were found, highlighting the occurrence of adaptation processes towards specific habitats.

Effect of the partial replacement of fish meal and oil by vegetable products on performance and quality traits of juvenile shi drum (Umbrina cirrosa L.)

Abstract A four-month growth trial was carried out in order to evaluate performance and quality traits of juvenile shi drum fed with two isonitrogenous and isoenergetic diets having different amounts of vegetable products (Vegetable diet vs. Control diet). Compared to the Control diet, the Vegetable diet was formulated by increasing the replacement of fish meal (14%) with soybean and cereal products, and fish oil (12%) with a mixture of vegetable oil. On June, 4 groups of 225 fish (2 replicates per dietary treatment) were sorted according to live weight and reared in fibreglass tanks over a four- month long experimental period. Fish were hand fed to apparent satiety. Offered feed, growth parameters and feed efficiency were recorded as productive performance. At the end of the trial (October) biometric, chemical and reological traits were examined to assess fish quality. The dietary treatments showed similar productive performance. The relatively high inclusion of vegetable sources led to a significant modification of body shape, mesenteric fat and viscera weight. Among quality traits, Vegetable diet-fed fish demonstrated a significantly lower whole body and fillet crude protein content. Yellowness value of the cooked fillet was significantly lower in the Control diet-fed fish, whereas fillet texture was similar. The results of this research showed that shi drum is a suitable candidate for Mediterranean marine aquaculture and its dietary formulation might include at least the amount of vegetable sources used in this trial.

Microbial dynamics during shelf-life of industrial Ricotta cheese and identification of a Bacillus strain as a cause of a pink discolouration.

Dairy products are perishable and have to be preserved from spoilage during the food chain to achieve the desired shelf-life. Ricotta is a typical Italian soft dairy food produced by heat coagulation of whey proteins and is considered to be a light and healthy product. The shelf-life of Ricotta could be extended, as required by the international food trade market; however, heat resistant microflora causes spoilage and poses issues regarding the safety of the product. Next-generation sequencing (NGS) applied to the Ricotta samples defined the composition of the microbial community in-depth during the shelf-life. The analysis demonstrated the predominance of spore-forming bacteria throughout the shelf-life, mostly belonging to Bacillus, Paenibacillus and Clostridium genera. A strain involved in spoilage and causing a pink discolouration of Ricotta was isolated and characterised as Bacillus mycoides/weihenstephanensis. This is the first report of a food discolouration caused by a toxigenic strain belonging to the Bacillus cereus group that resulted the predominant strain in the community of the defective ricotta. These results suggest that the processing of raw materials to eliminate spores and residual microflora could be essential for improving the quality and the safety of the product and to extend the shelf-life of industrial Ricotta.

Genotypic and phenotypic diversity of Pediococcus pentosaceus strains isolated from food matrices and characterisation of the penocin operon

Lactic acid bacteria (LAB) are widely used in the food industry. Pediococcus spp. belong to the LAB group and include several species that are essential for the quality of fermented food. Pediococcus pentosaceus is the species that is most frequently isolated from fermented food and beverages but its uncontrolled growth during food fermentation processes can contribute to undesired flavours. Hence, the characterisation of these bacteria at the strain level is of great importance for the quality of fermented products. Despite their importance, misidentification at the species level is common for members of the genus Pediococcus. To clarify the taxonomic relationships among strains, a multilocus sequencing approach was developed for the characterisation of a collection of 29 field strains, 1 type strain and 1 reference strain of P. pentosaceus isolated from food. These strains were also tested for several phenotypic properties of technological interest and for the production of bacteriocins. The chromosomal operon involved in the synthesis of the bacteriocin penocin was also investigated. The present study enabled a good genomic characterisation, identifying 17 sequence types, with an overview of phenotypic characteristics related to different technological abilities, and also provides a thorough characterisation of the operon involved in penocin production.