Lucia Aquilanti

Bacteria and yeast microbiota in milk kefir grains from different Italian regions

Kefir grains are a unique symbiotic association of different microrganisms, mainly lactic acid bacteria, yeasts and occasionally acetic acid bacteria, cohabiting in a natural polysaccharide and a protein matrix. The microbial composition of kefir grains can be considered as extremely variable since it is strongly influenced by the geographical origin of the grains and by the sub-culturing method used. The aim of this study was to elucidate the bacteria and yeast species occurring in milk kefir grains collected in some Italian regions by combining the results of scanning electron microscopy analysis, viable counts on selective culture media, PCR-DGGE and pyrosequencing. The main bacterial species found was Lactobacillus kefiranofaciens while Dekkera anomala was the predominant yeast. The presence of sub-dominant species ascribed to Streptococcus thermophilus, Lactococcus lactis and Acetobacter genera was also highlighted. In addition, Lc. lactis, Enterococcus sp., Bacillus sp., Acetobacter fabarum, Acetobacter lovaniensis and Acetobacter orientalis were identified as part of the cultivable community. This work further confirms both the importance of combining culture-independent and culture-dependent approaches to study microbial diversity in food and how the combination of multiple 16S rRNA gene targets strengthens taxonomic identification using sequence-based identification approaches.

Multidrug-Resistant Enterococci in Animal Meat and Faeces and Co-Transfer of Resistance from an Enterococcus durans to a Human Enterococcus faecium

Forty-eight isolates resistant to at least two antibiotics were selected from 53 antibiotic-resistant enterococci from chicken and pig meat and faeces and analysed for specific resistance determinants. Of the 48 multidrug-resistant (MDR) strains, 31 were resistant to two antibiotics (29 to erythromycin and tetracycline, 1 to erythromycin and vancomycin, 1 to vancomycin and tetracycline), 14 to three (erythromycin, tetracycline and vancomycin or ampicillin) and 3 to four (erythromycin, vancomycin, ampicillin and gentamicin). erm(B), tet(M), vanA and aac (6′)-Ie aph (2′′)-Ia were the antibiotic resistance genes most frequently detected. All 48 MDR enterococci were susceptible to linezolid and daptomycin. Enterococcus faecalis (16), Enterococcus faecium (8), Enterococcus mundtii (2) and Enterococcus gallinarum (1) were identified in meat, and E. faecium (13) and Enterococcus durans (13) in faeces. Clonal spread was not detected, suggesting a large role of gene transfer in the dissemination of antibiotic resistance. Conjugative transfer of resistance genes was more successful when donors were enterococcal strains isolated from faeces; co-transfer of vanA and erm(B) to a human E. faecium occurred from both E. faecium and E. durans pig faecal strains. These data show that multidrug resistance can be found in food and animal species other than E. faecium and E. faecalis, and that these species can efficiently transfer antibiotic resistance to human strains in inter-specific matings. In particular, the occurrence of MDR E. durans in the animal reservoir could have a role in the emergence of human enterococcal infections difficult to eradicate with antibiotics.

Investigation of the microbial ecology of Ciauscolo, a traditional Italian salami, by culture-dependent techniques and PCR-DGGE

The microbial ecology of 22 samples of commercially available Ciauscolo salami were investigated using a polyphasic approach, based on culture-dependent and -independent techniques. The viable counts of pathogen and hygiene indicator microorganisms highlighted the adequate application of good manufacturing practices, while the viable counts of the lactic acid bacteria, coagulase negative cocci, and yeasts showed dominance of the first of these microbial groups. Bacterial and fungal DNA were extracted directly from the salami and amplified by PCR, using two primer sets targeting the 16S and 28S rRNA genes, respectively. Denaturing gradient gel electrophoresis (DGGE) and sequencing of selected bands were used to investigate the microbial ecology of these Ciauscolo salami. The most frequently found bacterial species were Lactobacillus sakei and Lb. curvatus, while Debaryomyces hansenii was the prevalent yeast species detected. Cluster analysis of the DGGE profiles and calculation of biodiversity indices allowed the degree of microbial similarity across these salami to be determined.

Resident lactic acid bacteria in raw milk Canestrato Pugliese cheese

Aims:  Investigation of the autochthonous lactic acid bacteria (LAB) population of the raw milk Protected Designation of Origin Canestrato Pugliese cheese using phenotypic and genotypic methodologies.

Unpasteurised commercial boza as a source of microbial diversity

Boza is a cereal-based fermented beverage widely consumed in many countries of the Balkans. The aim of this study was to investigate the microbiota of three Bulgarian boza samples through a combination of culture-dependent and -independent methods with the long-term objective of formulating a multi-strain starter culture specifically destined for the manufacture of new cereal-based drinks. The isolation campaign for lactic acid bacteria (LAB) allowed the identification of Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus coryniformis, Lactobacillus buchneri, Pediococcus parvulus and members of the Lactobacillus casei group. Concerning yeasts, the following isolates were identified: Pichia fermentans, Pichia norvegensis, Pichia guilliermondii (synonym Meyerozyma guilliermondii) and Torulaspora spp. A high intra-species diversity was revealed by Randomly Amplified Polymorphic DNA (RAPD) analysis. In parallel, microbial DNA was directly extracted from the three boza samples, and portions of the rrn operons were analysed through Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The molecular fingerprinting partially confirmed the results of culturing. Among LAB, the species Weissella confusa, Weissella oryzae, Leuconostoc citreum, Lactococcus lactis, Pediococcus parvulus and Pediococcus ethanolidurans were detected together with members of the Lb. casei group. Among the yeasts, the species P. fermentans, M. guilliermondii, Galactomyces geotrichum and Geotrichum fragrans were found. The overall results confirmed boza as having a rich and heterogeneous biodiversity both in terms of species and genetically diverse strains, thus encouraging its exploitation for the isolation and future technological characterisation of cultures to be selected for the manufacture of innovative cereal-based drinks.

An eight-year report on the implementation of HACCP in a university canteen: impact on the microbiological quality of meals

An investigation aimed at assessing the microbiological quality of meals consumed at a university canteen after implementation of the HACCP system and personnel training was carried out. Cooked and warm-served products (74 samples), cooked and cold-served products (92 samples) and cold gastronomy products (63 samples) sampled from 2000 to 2007 underwent microbiological analyses. All the samples were tested for: Samonella spp., Listeria monocytogenes, total mesophilic aerobes, coliforms, Escherichia coli, Staphylococcus aureus, Bacillus cereus, and sulphite-reducing clostridia. The microbiological contamination of work surfaces (tables, tablewares, cutters, ladles, slicing machines, wash-basins, etc.), hands and white coats of members of the canteen staff was also assessed. The microbiological results clearly demonstrated the success of the HACCP plan implementation, through a general improvement of the hygiene conditions of both meals and work surfaces.

Recent investigations and updated criteria for the assessment of antibiotic resistance in food lactic acid bacteria

The worldwide use, and misuse, of antibiotics for about sixty years in the so-called antibiotic era, has been estimated in some one to ten million tons, a relevant part of which destined for non-therapeutic purposes such as growth promoting treatments for livestock or crop protection. As highly adaptable organisms, bacteria have reacted to this dramatic change in their environment by developing several well-known mechanisms of antibiotic resistance and are becoming increasingly resistant to conventional antibiotics. In recent years, commensal bacteria have become a cause of concern since they may act as reservoirs for the antibiotic resistance genes found in human pathogens. In particular, the food chain has been considered the main route for the introduction of animal and environment associated antibiotic resistant bacteria into the human gastrointestinal tract (GIT) where these genes may be transferred to pathogenic and opportunistic bacteria. As fundamental microbial communities in a large variety of fermented foods and feed, the anaerobe facultative, aerotolerant lactic acid bacteria (LAB) are likely to play a pivotal role in the resistance gene exchange occurring in the environment, food, feed and animal and human GIT. Therefore their antibiotic resistance features and their genetic basis have recently received increasing attention. The present article summarises the results of the latest studies on the most typical genera belonging to the low G + C branch of LAB. The evolution of the criteria established by European regulatory bodies to ensure a safe use of microorganisms in food and feed, including the assessment of their antibiotic resistance is also reviewed.

The microbiota of marketed processed edible insects as revealed by high-throughput sequencing

Entomophagy has been linked to nutritional, economic, social and ecological benefits. However, scientific studies on the potential safety risks in eating edible insects need to be carried out for legislators, markets and consumers. In this context, the microbiota of edible insects deserves to be deeply investigated. The aim of this study was to elucidate the microbial species occurring in some processed marketed edible insects, namely powdered small crickets, whole dried small crickets (Acheta domesticus), whole dried locusts (Locusta migratoria), and whole dried mealworm larvae (Tenebrio molitor), through culture-dependent (classical microbiological analyses) and -independent methods (pyrosequencing). A great bacterial diversity and variation among insects was seen. Relatively low counts of total mesophilic aerobes, Enterobacteriaceae, lactic acid bacteria, Clostridium perfringens spores, yeasts and moulds in all of the studied insect batches were found. Furthermore, the presence of several gut-associated bacteria, some of which may act as opportunistic pathogens in humans, were found through pyrosequencing. Food spoilage bacteria were also identified, as well as Spiroplasma spp. in mealworm larvae, which has been found to be related to neurodegenerative diseases in animals and humans. Although viable pathogens such as Salmonella spp. and Listeria monocytogenes were not detected, the presence of Listeria spp., Staphylococcus spp., Clostridium spp. and Bacillus spp. (with low abundance) was also found through pyrosequencing. The results of this study contribute to the elucidation of the microbiota associated with edible insects and encourage further studies aimed to evaluate the influence of rearing and processing conditions on that microbiota.

Selection of sourdough lactobacilli with antifungal activity for use as biopreservatives in bakery products.

Two hundred and sixteen LAB cultures from sourdoughs and dough for bread and panettone production were screened for in vitro antifungal properties against three indicator cultures ascribed to Aspergillus japonicus , Eurotium repens , and Penicillium roseopurpureum , isolated from bakery environment and moldy panettone. Nineteen preselected isolates were subjected to minimum inhibitory concentration determination against the indicator cultures. Sourdoughs prepared with the two most promising strains, identified as Lactobacillus rossiae LD108 and Lactobacillus paralimentarius PB127, were characterized. The sourdough extracts were subjected to HPLC analysis coupled with a microtiter plate bioassay against A. japonicus to identify the active fractions. MALDI-TOF MS analysis revealed the occurrence of a series of peptides corresponding to wheat α-gliadin proteolysis fragments in the active fraction from L. rossiae LD108 sourdough. The ability to prevent mold growth on bread was demonstrated for both strains, whereas L. rossiae LD108 also inhibited mold growth on panettone.

Lactic acid bacteria and yeasts from wheat sourdoughs of the Marche Region

The need for a greater diversification of baked products has given rise to the on-going search for yeast and lactic acid bacteria (LAB) strains with optimal baking potential. Thirty-six yeasts and 118 LAB, isolated from nine type I sourdoughs that were sampled in bakeries located in the Marche region (central Italy), were molecularly and phenotypically characterized. The polyphasic approach used revealed the biodiversity of the microbial communities investigated and two yeasts and ten LAB cultures with the potential to be used in sourdough bread-making processes were identified.