Filomena Montemurro

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.

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.

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.

Minimum bactericidal concentration of phenols extracted from oil vegetation water on spoilers, starters and food-borne bacteria

The aim of the study was to assess the in vitro effect of phenols extracted from oil vegetation water (PEOW) on several food-borne strains. Antibacterial activity of PEOW was based on the minimum bactericidal concentration (MBC) on microtitre assay. The taxa tested were: Staphylococcus (n. 5), Listeria (n. 4), Escherichia (n. 2), Salmonella (n. 1), Pseudomonas (n. 3), Lactobacillus (n. 2) and Pediococcus (n. 1). S. aureus and L. monocytogens showed the lowest level of resistance to PEOW (MBC=1.5-3 mg/mL). In contrast, the Gram negative strains (e.g. S. Typhimurium and Pseudomonas spp.) were in some cases unaffected by the tested doses and the MBCs ranged between 6 to 12 mg/mL. Starter cultures were dramatically reduced on growth (e.g. Staphylococcus xylosus; 0.75 mg/mL MBC). The thresholds for pathogenic strains could be considered for further applications of PEOW in food models (e.g. shelf life or challenge test studies).

Storage test on apple juice after ultrasound treatment

Apple juice, for its sensory and nutritional qualities, is consumed by people of all ages. Apples are an excellent source of several phenolic compounds and the presence of polyphenols is recognized for their health promoting antioxidant properties. Thermal pasteurization of fruit juices is the conventional method used for their preservation. Therefore, this constitutes the most extensively available methods for the inactivation of microorganisms in fruit juices but it causes side effects on their flavour and nutritional quality. Consumers tend to prefer recently extracted juices with fresh taste and minimal flavor or vitamin losses. To meet consumers’ demand, among the novel technologies that involve non-thermal processes, power ultrasound have been investigated as an alternative to conventional heat treatments. Objective of this study was to evaluate the effectiveness of the use of ultrasound in an attempt to maintain the organoleptic characteristics typical of a natural apple juice. In particular, it was evaluated the action on the microflora residing and shelf life of the product through microbiological and sensory analyses. Juice treated with ultrasound highlighted a reduction of aerobic mesophilic counts and psychrophilic bacteria respectively about 3 and 5 log CFU/mL and an enhanced yeast growth. The general opinion expressed by the panelist was in favour of the sonicated juice. This preliminary study showed that non-thermal methods such as power ultrasound technology may give new opportunities to develop fresh-like apple juice.