EVALUATION OF ANTIBIOTIC RESISTANCE PROFILES OF HALOBACTERIA ISOLATED FROM THE FOOD CHAIN

L’insorgenza e la diffusione dell’antibiotico resistenza sta diventando un problema a livello mondiale. Molti sono gli ambienti in cui può avvenire tale diffusione, ma una delle principali vie di trasmissione passa attraverso la catena alimentare. Infatti, l’utilizzo di sostanze antimicrobiche è largamente diffuso negli allevamenti di animali ad uso alimentare e in agricoltura. In particolare, negli allevamenti gli antibiotici non solo vengono usati per trattare eventuali patologie, ma anche come profilassi e come promotori di crescita. Di conseguenza, questo uso a volte sconsiderato ha portato all’insorgenza di batteri resistenti a tali sostanze. Un ruolo fondamentale nella trasmissione e diffusione di tali resistenze a livello alimentare è svolto da batteri non patogeni che sono parte del naturale microbiota degli alimenti. Questi microorganismi infatti, pur non essendo essi stessi nocivi per l’uomo, possono fungere da reservoir di antibiotico resistenze per eventuali batteri patogeni. I batteri che generalmente svolgono questo ruolo sono i batteri lattici. Per questo motivo molto importante è stato identificare e studiare l’antibiotico resistenza anche di tali microorganismi. Negli ultimi anni, tuttavia, c’è stato un crescente interesse per un’altra classe di microorganismi, chiamata Haloarchaea o alobatteri o archaea alofili, poiché la loro presenza è stata rilevata in alimenti particolarmente salati. Dal momento che in letteratura ci sono pochi lavori che studiano i profili di antibiotico resistenza di tali microorganismi e, comunque, tali profili non sono stati studiati su un numero significativo di microorganismi appartenenti alla stessa specie, il presente lavoro di tesi è volto a definire il profilo di antibiotico resistenza del capostipite degli archaea alofili, che è l’Halobacterium salinarum, verificare se ci sono ceppi che presentano antibiotico resistenze e controllare se tali resistenze possono essere trasferite a batteri patogeni.Antimicrobial resistance is now widely acknowledged as a major global public health challenge. There are many environments through which the transmission and diffusion of antibiotic resistance could happen, but one of the main routes of transmission is the food chain. As a matter of fact, antibiotic use is widely spread in animal husbandry and in agriculture. In particular, in animal husbandry antimicrobials have been used both for therapeutic reasons and as growth promoters. As a consequence, a selective pressure on pathogenic and commensal bacteria of animal origin has been exerted during the time, leading to the onset of microorganisms resistant to such compounds. A pivotal role in the spread in the food chain of antibiotic resistance has been played by non-pathogenic bacteria present in food. These microorganisms are not harmful for humans, but they could represent a reservoir of antibiotic resistance for foodborne pathogenic bacteria. Usually lactic acid bacteria play this role, since they are present in all fermented food. For this reason, the antibiotic resistance profile of lactic acid bacteria has been assessed. In recent years, another class of microorganisms called halophilic archaea have raised an increasing scientific interest, since they have been found in the human intestinal mucosa as well as in foods such as salted codfish and fermented Asiatic seafood. As a few papers have studied the antibiotic resistance profiles of halophilic archaea, and the only present do not consider a statistically significant number of microorganisms belonging to the same species, the aim of the present work is to define the antibiotic resistance profile of the major exponent of halophilic archaea, named Halobacterium salinarum, and consequently to verify if some strains present antibiotic resistances and if they can transfer these resistances to bacteria present in the food chain

Swine granulosa cells show typical endothelial cell characteristics

Abstract. Granulosa cells, which belong to the somatic compartment of the ovarian follicle, are actively involved as endocrine cells in follicle growth. Recently, it has been proposed that these cells are not terminally differentiated and possess multipotency. Therefore, we cultured swine granulosa cells in specific endothelial cell culture medium (EBM-2) and phenotypic and functional characteristics of endothelial cells were assessed. The collected data suggest that these endocrine cells can also behave as endothelial cells therefore potentially contributing to follicular angiogenesis, a crucial process in follicle growth and selection

Towards the definition of minimal inhibitory concentrations for Halobacterium salinarum

Archaea are prokaryotic organisms thriving mostly at extreme environments and sharing characteristics with both bacteria and eukaryotes. Within the Archaea domain, the Halobacteriaceae family comprises species that are mainly found in solar salterns, marine environments and salted food products. A major representative of this group is Halobacterium salinarum. Recently, these microorganisms have raised an increasing scientific interest, since they have been found in both food and human intestinal mucosa. Very little is known about their antibiotic resistances, and it is thus important to establish microbiological breakpoints for Halobacteriaceae, as done in the past for bacteria. At present only few works have studied antibiotic resistance in halophilic Archaea, but the Minimal Inhibitory Concentrations (MICs) proposed by these studies can’t be considered acceptable since they didn’t use at least 50 different strains for each species, as the European Committee on Antimicrobial Susceptibility (EUCAST) requires. Fifty-two different strains of Halobacterium salinarum were isolated and characterised by RAPD (Random Amplification of Polymorphic DNA) and 16S rRNA analyses within a study on the microbial ecology of animal casings. We assessed the microbiological breakpoints of these isolates and other three collection strains against anisomycin, ciprofloxacin, clindamycin, erythromycin, novobiocin, rifampicin and trimethoprim. While for rifampicin, ciprofloxacin and trimethoprim the MICs previously found have been confirmed, for the other antibiotics we obtained for clindamycin, novobiocin and anisomycin MICs lower than the reported ones, while for erythromycin they were higher. In particular, we observed that H. salinarum is mainly susceptible to antibiotics that inhibit DNA replication rather than those which interact with protein synthesis. High variability among strains was mainly found with novobiocin. This work provides new insights about MICs for H. salinarum, a strain that is often found along the food chain, and which can be a first model for the assessment of antibiotic resistance profiles in Archaea

Analysis of the cross-talk between maize and Fusarium verticillioides revealed by RNA-Sequencing.

Fusarium verticillioides is a plant pathogen able to produce fumonisin in maize kernels. To clarify the molecular processes undergoing in maize upon infection, the expression profiles of resistant and susceptible genotypes after F. verticillioides inoculation have been characterized using RNA-Seq technology. More than 100 million sequence reads were generated for condition (inoculated/uninoculated). The sequence reads were analyzed to measure gene expression levels, to detect alternative splicing events and single nucleotide polymorphisms. We observed 2,296 and 2,578 differentially expressed genes (DEGs) 72 hours after inoculation for the resistant and susceptible genotypes, respectively, of which 1018 were in common and showed 5,342 SNPs variants. About 320,000 and 175,000 reads mapped on Fusarium genome in the susceptible and resistant genotypes, respectively, and 129 fungal genes were differentially regulated in both lines. The identification of plant DEGs that interact with fungus will produce useful tools for the identification of candidate genes, the development of molecular markers and their use for selection of resistant maize genotypes by means of marker assisted selection

General and genotype-specific transcriptional responses to Fusarium ear rot in resistant and susceptible maize genotypes.

Fusarium ear rot caused by Fusarium verticillioides is a prevalent disease in maize which can severely reduce graind yields and quality, due to contamination of infected kernels with mycotoxins. Natural sources of resistance from maize genotypes are used in conventional breeding approaches, but the signals and effectors involved in resistance in this important crop species are not well understood. Early transcriptional changes associated with F. verticillioides infection were analysed in resistant CO441 and susceptible CO354 maize genotypes using RNA-Sequencing technology. Transcript levels were measured at 72 hours post-inoculation (hpi), reflecting the time point immediately preceding the onset of resistance in CO441, as determined by the absolute quantification of the fungal β-tubulin2 gene. More than 100 million sequence reads were generated for condition (inoculated/uninoculated). The sequence reads were analyzed to measure gene expression levels and determine single nucleotide polymorphisms. We observed 2,551 transcripts with statistically significant differential expression before inoculation by comparing uninoculated control samples. Overall, a more induced expression of genes was found in the CO441 genotype, distributed in all functional classes, and in particular a high percentage of differentially expressed genes was detected for the category secondary metabolism. Furthermore, a total of 2,250 and 2,442 transcripts were differentially regulated after F. verticillioides inoculation in resistant and susceptible genotypes, respectively. The genes in common were 1,028 genes and showed 5,342 SNPs variants, suggesting a certain variability in the defense responses of the two genotypes. Also at 72 hpi, secondary metabolism was the category where the most interesting differences between the two genotypes were observed to a greater extent. Pathways related to phenylalanine, tyrosine and tryptophan biosynthesis (e.g. anthranilate synthase, shikimate kinase), flavonoid biosynthesis (e.g. chalcone synthase) and lignin biosynthesis (e.g. cinnamyl-alcohol dehydrogenase) appeared to be strongly influenced by infection in the CO441 genotype with up to 11-fold induction. Response to stress and resistance categories followed. In addition, transcriptional modulation affected signal transduction, including calcium signaling, ethylene signaling, MAP kinases, receptor-like kinases, and numerous transcription factors. Even though all these genes were well represented in both genotypes, it was observed that on the whole CO441 mounts a stronger gene induction, confirming its strengthening also in the specific response to inoculation. The identification of plant candidate resistance genes that interact with fungus could be exploited in future biotechnological approaches to increase disease resistance in susceptible maize genotypes

Comparative genomics of Halobacterium salinarum strains isolated from salted foods reveals protechnological genes for food applications

Archaeal cell factories are becoming of great interest given their ability to produce a broad range of value-added compounds. Moreover, the Archaea domain often includes extremophilic microorganisms, facilitating their cultivation at the industrial level under nonsterile conditions. Halophilic archaea are studied for their ability to grow in environments with high NaCl concentrations. In this study, nine strains of Halobacterium salinarum were isolated from three different types of salted food, sausage casings, salted codfish, and bacon, and their genomes were sequenced along with the genome of the collection strain CECT 395. A comparative genomic analysis was performed on these newly sequenced genomes and the publicly available ones for a total of 19 H. salinarum strains. We elucidated the presence of unique gene clusters of the species in relation to the different ecological niches of isolation (salted foods, animal hides, and solar saltern sediments). Moreover, genome mining at the single-strain level highlighted the metabolic potential of H. salinarum UC4242, which revealed the presence of different protechnological genes (vitamins and myo-inositol biosynthetic pathways, aroma- and texture-related features, and antimicrobial compounds). Despite the presence of genes of potential concern (e.g., those involved in biogenic amine production), all the food isolates presented archaeocin-related genes (halocin-C8 and sactipeptides)

From gut to food and back to gut: bacterial diversity in animal casings used in the production of dry fermented sausages

Introduction Typical dry fermented sausages have been produced for centuries using natural casings, which are portions of animal intestines derived from slaughtering. Natural casings are very important components of many traditional meat fermented products, and they represent a peculiar case study of gut-derived environments that are used for the production of edible fermented foods. Materials & Methods In the present study we investigated by means of culture-dependent methods and Illumina high-throughput sequencing of 16S rRNA amplicons the bacterial ecology of hog, cow and ovine casings at different stages of their preparation for sausages production. Results Several strains of Staphylococcus, Lactobacillus, Bifidobacterium, Vagococcus and Clostridium were counted in significant amounts, isolated and characterized at phylogenetic level. High-throughput sequencing analyses revealed a highly diverse bacterial diversity, which differed strongly between casings of different animal species. The technological processes involved in the preparation for casing had also a strong impact on the casings bacterial ecology, with a significant reduction of undesired microorganisms, and an increase in the proportion of lactobacilli and staphylococci. Conclusions Overall results indicate that natural casings are an important source of several bacterial species whose role both in the fermentation and in the microbiological properties of the final products has been underestimated

Animal casings as a source of microorganisms involved in meat fermentation: evidence from culture-based and high-throughput molecular methods

Natural casings are portions of animal intestines that have been used for centuries in the manufacturing of many typical dry-fermented sausages. Because of their intestinal origin, casings have a high microbial load, but their possible role as inoculants of microorganisms that can play a role in the ripening of dry-fermented sausages has not been investigated yet in detail. Here, we analyzed by means of culture-dependent methods and Illumina high- throughput sequencing of 16S rRNA amplicons the bacterial ecology of hog, cow and ovine casings at different stages of their preparation for sausages production. We also compared the bacterial ecology of casings, meat mixtures and sausages at different ripening stages in the production chains of Salame Mantovano, a typical Italian dry-fermented sausage. Culture-based methods relied on the isolation and characterization of strains on different typical media, while for the molecular methods multi-million reads were originated and analyzed after amplification and Illumina MiSeq sequencing of 16S rRNA amplicons. From the animal casings, several strains of Staphylococcus, Lactobacillus, Bifidobacterium, Vagococcus and Clostridium were counted in significant amounts, isolated and characterized at phylogenetic level. High-throughput sequencing analyses revealed a high bacterial diversity, which differed strongly between casings of different animal species. It was also found that the technological processes had a strong impact on the casings bacterial ecology, with a significant reduction of undesired microorganisms, and an increase in the proportion of lactobacilli and staphylococci. Finally, the analyses on the production chain of Salame Mantovano revealed that several strains found in the casings before stuffing were also detected in the final ripened products, thus confirming our main hypothesis. This work shows the importance of the use of natural casings in the manufacturing of typical dry-fermented products, and highlights the role of high-throughput sequencing technologies as powerful tools to gain a better comprehension of food fermentations

High-throughput asssessment of bacterial ecology in hog, cow and ovine casings used in sausages production

Natural casings derived from different intestine portions have been used for centuries in the production of fresh and dry-fermented sausages. Here we analysed by means of culture-dependent methods and Illumina high-throughput sequencing of 16S rRNA amplicons the bacterial ecology of hog, cow and ovine casings at different stages of their preparation for sausages production. Several strains of Staphylococcus, Lactobacillus, Bifidobacterium, Vagococcus and Clostridium were counted, isolated and characterised at phylogenetic level. High-throughput sequencing analyses revealed a high bacterial diversity, which differed strongly between casings of different animal species. The technological processes involved in the preparation for casing had also a strong impact on the casings bacterial ecology, with a significant reduction of undesired microorganisms, and an increase in the proportion of lactobacilli and staphylococci. Natural casings were demonstrated to be complex ecological environments, whose role as microbiological inoculants in the production of sausages should not be underestimated

Isolation and characterization of lactic acid bacteria and yeasts from fermented sorghum flour

Sorghum (Sorghum vulgare Pers.) is a crop of great interest due to its high yield, its tolerance of adverse agronomic conditions that are becoming more and more frequent for the climate change, and its nutritional quality. In particular, the peculiar organization of the macronutrients (starch and proteins) of this cereal has led to the development of rudimental technologies to exploit its high value in the countries where its use is widely spread for the production of both food and beverages. Nevertheless, in Italy, it is primarily intended for zootechnical use and its application in human nutrition is still limited by cultural constrains. One of the most interesting characteristics of sorghum is the lack of gluten, which makes this cereal an eligible ingredient for coeliac people food. On the other hand, the lack of gluten limits the application of sorghum flour in the bakery industry. For this reason, after spontaneous fermentation of sorghum flour, we isolated and characterized lactic acid bacteria presenting useful characteristics able to improve the technological handling of this ingredient, in order to make sorghum flour usable for bakery products. Moreover, we have evaluated the pH and the Titratable Acidity (TTA) throughout the fermentation process. Regarding the sorghum flour microbiota, we have identified different strains of Weissella spp for the bacterial part, together with Pichia spp and Hanseniaspora spp for the yeast counterpart