Expression of Caseicin from Lacticaseibacillus casei and Lacticaseibacillus zeae Provides Insight into Antilisterial Class IIa Bacteriocins.

In this study, an in silico screening approach was employed to mine potential bacteriocin clusters in genome-sequenced isolates of Lacticaseibacillus zeae UD 2202 and Lacticaseibacillus casei UD 1001. Two putative undescribed bacteriocin gene clusters (Cas1 and Cas2) closely related to genes encoding class IIa bacteriocins were identified. No bacteriocin activity was recorded when cell-free supernatants of strains UD 2202 and UD 1001 were tested against Listeria monocytogenes. Genes encoding caseicin A1 (casA1) and caseicin A2 (casA2) were heterologously expressed in Escherichia coli BL21 (DE3) using the nisin leader peptide cloned in-frame to the C-terminal of the green fluorescent gene (mgfp5). Nisin protease (NisP) was used to cleave caseicin A1 (casA1) and caseicin A2 (casA2) from GFP-Nisin leader fusion proteins. Both heterologously expressed peptides (casA1 and casA2) inhibited the growth of L. monocytogenes, suggesting that casA1 and casA2 are either silent in the wild-type strains or are not secreted in an active form. The minimum inhibitory concentration (MIC) of casA1 and casA2, determined using HPLC-purified peptides, ranged from < 0.2 µg/mL to 12.5 µg/mL when tested against Listeria ivanovii, Listeria monocytogenes, and Listeria innocua, respectively. A higher MIC value (25 µg/mL) was recorded for casA1 and casA2 when Enterococcus faecium HKLHS was used as the target. The molecular weight of heterologously expressed casA1 and casA2 is 5.1 and 5.2 kDa, respectively, as determined with tricine-SDS-PAGE. Further research is required to determine if genes within Cas1 and Cas2 render immunity to other class IIa bacteriocins.

Cyberlindnera fabianii, an Uncommon Yeast Responsible for Gluten Bread Spoilage.

A single strain of yeast was isolated from industrial gluten bread (GB) purchased from a local supermarket. This strain is responsible for spoilage consisting of white powdery and filamentous colonies due to the fragmentation of hyphae into short lengths (dust-type spots), similar to the spoilage produced by chalk yeasts such as Hyphopichia burtonii, Wickerhamomyces anomalus and Saccharomycopsis fibuligera. The isolated strains were identified initially by traditional methods as Wickerhamomyces anomalus, but with genomic analysis, they were definitively identified as Cyberlindnera fabianii, a rare ascomycetous opportunistic yeast species with low virulence attributes, uncommonly implicated in bread spoilage. However, these results demonstrate that this strain is phenotypically similar to Wi. anomalus. Cy. fabianii grew in GB because of its physicochemical characteristics which included pH 5.34, Aw 0.97 and a moisture of about 50.36. This spoilage was also confirmed by the presence of various compounds typical of yeasts, derived from sugar fermentation and amino acid degradation. These compounds included alcohols (ethanol, 1-propanol, isobutyl alcohol, isoamyl alcohol and n-amyl alcohol), organic acids (acetic and pentanoic acids) and esters (Ethylacetate, n-propil acetate, Ethylbutirrate, Isoamylacetate and Ethylpentanoate), identified in higher concentrations in the spoiled samples than in the unspoiled samples. The concentration of acetic acid was lower only in the spoiled samples, but this effect may be due to the consumption of this compound to produce acetate esters, which predominate in the spoiled samples.

Genotypic and phenotypic profiling of 127 Legionella pneumophila strains: Insights into regional spread.

Given the recent global surge in Legionnaires' disease cases, the monitoring of Legionella pneumophila becomes increasingly crucial. Epidemiological cases often stem from local outbreaks rather than widespread dissemination, emphasizing the need to study the characteristics of this pathogen at a local level. This study focuses on isolates of L. pneumophila in the Italian region of Friuli Venezia Giulia to assess specific genotype and phenotype distribution over time and space. To this end, a total of 127 L. pneumophila strains isolated between 2005 and 2017 within national surveillance programs were analysed. Rep-PCR, RAPD, and Sau-PCR were used for genotypic characterization, while phenotypic characterization was conducted through fatty acids analysis. RAPD and Sau-PCR effectively assessed genetic characteristics, identifying different profiles for the isolates and excluding the presence of clones. Although Sau-PCR is rarely used to analyse this pathogen, it emerged as the most discriminatory technique. Phenotypically, hierarchical cluster analysis categorized strains into three groups based on varying membrane fatty acid percentages. However, both phenotypic and genotypic analyses revealed a ubiquitous profile distribution at a regional level. These results suggest an absence of correlations between strain profiles, geographical location, and isolation time, indicating instead high variability and strain dissemination within this region.

Investigating Safety and Technological Traits of a Leading Probiotic Species: Lacticaseibacillus paracasei.

Lacticaseibacillus spp. are genetically close lactic acid bacteria species widely used in fermented products for their technological properties as well as their proven beneficial effects on human and animal health. This study, the first to include such a large collection of heterogeneous isolates (121) obtained from international collections belonging to Lacticaseibacillus paracasei, aimed to characterize the safety traits and technological properties of this important probiotic species, also making comparisons with other genetically related species, such as Lacticaseibacillus casei and Lacticaseibacillus zeae. These strains were isolated from a variety of heterogeneous sources, including dairy products, sourdoughs, wine, must, and human body excreta. After a preliminary molecular characterization using repetitive element palindromic PCR (Rep-PCR), Random Amplification of Polymorphic DNA (RAPD), and Sau-PCR, particular attention was paid to safety traits, evaluating antibiotic resistance profiles, biogenic amine (BA) production, the presence of genes related to the production of ethyl carbamate and diaminobenzidine (DAB), and multicopper oxidase activity (MCO). The technological characteristics of the strains, such as the capability to grow at different NaCl and ethanol concentrations and different pH values, were also investigated, as well as the production of bacteriocins. From the obtained results, it was observed that strains isolated from the same type of matrix often shared similar genetic characteristics. However, phenotypic traits were strain-specific. This underscored the vast potential of the different strains to be used for various purposes, from probiotics to bioprotective and starter cultures for food and feed production, highlighting the importance of conducting comprehensive evaluations to identify the most suitable strain for each purpose with the final aim of promoting human health.

Leuconostoc gelidum Is the Major Species Responsible for the Spoilage of Cooked Sausage Packaged in a Modified Atmosphere, and Hop Extract Is the Best Inhibitor Tested.

Cooked sausages packaged in a modified atmosphere (MAP: 20% CO2, 70% N2, <0.2% O2) with evident yellow stains were analyzed. The aims of this work were to study the microbial cause of the spoilage and to evaluate different antimicrobial compounds to prevent it. Leuconostoc gelidum was identified as the primary cause of the yellow coating in spoiled cooked sausage, as confirmed by its intentional inoculation on slices of unspoiled sausage. Leuconostoc gelidum was the main bacteria responsible for the yellow coating in spoiled cooked sausage, as confirmed by its intentional inoculation on slices of unspoiled sausage. The yellow color was also evident during growth in the model system containing cooked sausage extract, but the colonies on MRS agar appeared white, demonstrating that the food substrate stimulated the production of the yellow pigment. The spoilage was also characterized by different volatile compounds, including ketones, ethanol, acetic acid, and ethyl acetate, found in the spoiled cooked sausage packages. These compounds explained the activity of Leuc. gelidum because they are typical of heterofermentative LAB, cultivated either on food substrates or in artificial broths. Leuc. gelidum also produced slight swelling in the spoiled packages. The efficacy of different antimicrobials was assessed in model systems composed of cooked sausage extract with the antimicrobials added at food product concentrations. The data showed that sodium lactate, sodium acetate, and a combination of sodium lactate and sodium diacetate could only slow the growth of the spoiler-they could not stop it from occurring. Conversely, hop extract inhibited Leuc. gelidum, showing a minimal inhibitory concentration (MIC) of approximately 0.008 mg CAE/mL in synthetic broth and 4 mg CAE/kg in cooked sausage slices. Adding hop extract at the MIC did not allow Leuc. gelidum growth and did not change the sensorial characteristics of the cooked sausages. To our knowledge, this is the first report of the antimicrobial activities of hop extracts against Leuc. gelidum either in vitro or in vivo.

Draft genome sequences from 127 Legionella spp. strains isolated in water systems linked to legionellosis outbreaks.

Legionnaires' disease is a severe form of pneumonia caused by Legionella spp. bacteria. According to the European Centre for Disease Prevention and Control, problems related to this pathogen showed a significant surge in recent years, making its monitoring critical.

Ancient Roman bacterium against current issues: strain Aquil_B6, Paenisporosarcina quisquiliarum, or Psychrobacillus psychrodurans?

IMPORTANCE: Since 1988, through the United States government's founding, the National Center for Biotechnology Information (NCBI) has provided an invaluable service to scientific advancement. The universality and total freedom of use if on the one hand allow the use of this database on a global level by all researchers for their valuable work, on the other hand, it has the disadvantage of making it difficult to check the correctness of all the materials present. It is, therefore, of fundamental importance for the correctness and ethics of research to improve the databases at our disposal, identifying and amending the critical issues. This work aims to provide the scientific community with a new sequence for the type strain Paenisporosarcina quisquiliarum SK 55 and broaden the knowledge of the Psychrobacillus psychrodurans species, in particular, considering the ancient strain Aquil_B6 found in an ancient Roman amphora.

Organic vs. conventional: impact of cultivation treatments on the soil microbiota in the vineyard.

The aim of this study was to compare the effects of two vineyard management practices on the soil and its associated microbiota. The experiments were conducted in two adjacent plots, one completely organically managed and the other conventionally managed in terms of phytosanitary treatments but fertilized with organic amendments. The chemical soil analyses were correlated to the prokaryotic and fungal communities, which were studied using the metabarcoding technique. The main difference between the two treatments was a significantly higher amount of Cu in the organic managed vineyard soil, while conventional managed soil presented higher concentration of Na and Mg and was also associated with higher pH values. Despite these differences, no significant diversities were observed on soil biodiversity and microbial composition considering alpha and beta diversity metrics. However, the percentages of some phyla analyzed individually differed significantly between the two managements. Analyzing the metabolisms of these phyla, it was discovered an increment of species correlated to soils with higher organic matter content or land not used for agricultural purposes in the organic treated soil. The findings indicate that, despite the use of copper-based phytosanitary products, there was no degradation and loss of biodiversity in the organic soil microbial population compared to conventional management with the same type of fertilization, and the observed microbial population was more similar to that of natural soils.

Draft genome sequences of 14 Lacticaseibacillus spp. strains, representatives of a collection of 200 strains.

Lactobacilli have a fundamental role in the food industry as starters and probiotics, therefore, requiring special attention concerning food safety. In this work, 14 strains selected accordingly to their genetic fingerprint and physiologic characteristics are presented as representatives of a collection of 200 strains.

Microbial Spoilage of Traditional Goose Sausages Produced in a Northern Region of Italy.

Recently, during the ripening of goose sausage, a defect consisting of ammonia and vinegar smell was noticed. The producer of the craft facility, located in Lombardia, a Northern region of Italy, asked us to identify the cause of that defect. Therefore, this study aimed to identify the potential responsible agents for the spoilage of this lot of goose sausages. Spoilage was first detected by sensory analysis using the "needle probing" technique; however, the spoiled sausages were not marketable due to the high ammonia and vinegar smell. The added starter culture did not limit or inhibit the spoilage microorganisms, which were represented by Levilactobacillus brevis, the predominant species, and by Enterococcus faecalis and E. faecium. These microorganisms grew during ripening and produced a large amount of biogenic amines, which could represent a risk for consumers. Furthermore, Lev. brevis, being a heterofermentative lactic acid bacteria (LAB), also produced ethanol, acetic acid, and a variation in the sausage colour. The production of biogenic amines was confirmed in vitro. Furthermore, as observed in a previous study, the second cause of spoilage can be attributed to moulds which grew during ripening; both the isolated strains, Penicillium nalgiovense, added as a starter culture, and P. lanosocoeruleum, present as an environmental contaminant, grew between the meat and casing, producing a large amount of total volatile nitrogen, responsible for the ammonia smell perceived in the ripening area and in the sausages. This is the first description of Levilactobacillus brevis predominance in spoiled goose sausage.

Microbial and Physico-Chemical Characterization of Cold Smoked Sea Bass (Dicentrarchus labrax), a New Product of Fishery.

The aim of this study was to investigate the microbial and physico-chemical characteristics of cold smoked sea bass (CSSB), a novel italian fish product. The microbiological analyses showed the presence of bacterial contamination from the raw material, the environment, and the production process. The microbial spoilage population was dominated by lactic acid bacteria (LAB) associated with Gram-negative fermenting bacteria, including Photobacterium phosphoreum and psychrotrophic Enterobacteriaceae. Brochotrix thermospacta and Aeromonas spp. were also present; in contrast, mould and yeast were not detected (<2 CFU/g). High levels (6-7 log CFU/g) of LAB and total bacteria count (TBC) were observed from day 45 of storage; however, their presence does not seem to have influenced the total volatile basic nitrogen (TVB-N), which always remained below 35 mg N/100 g. Consequently, the product is acceptable until day 60 of storage, considering that the malonaldehyde index (TBARS) was lower than 6.5 nmol/g. Pathogenic bacteria such as Salmonella spp. and Listeria monocytogenes were not detected. Currently, there is a growing demand for seafood due to its high quality and nutritional value. Cold smoked sea bass offers a source of macro- and micronutrients essential for the proper functioning of the human body. It is also rich in protein and omega-3 fatty acids. The WHO and FAO evaluated the benefits and risks and concluded that there is convincing evidence of health benefits from fish consumption, such as a reduction in the risk of heart failure and improved neurodevelopment in infants and young children when fish is consumed by the mother before and during pregnancy. The CSSB analysed in this study demonstrated to have health benefits due to long-chain omega-3 PUFAs and other nutrients, such as proteins, minerals, and vitamin D, which are sometimes difficult to obtain from other sources. The results show that CSSB has a high nutritional value and excellent microbial quality.

Legionella anisa or Legionella bozemanii? Traditional and molecular techniques as support in the environmental surveillance of a hospital water network.

Understanding the actual distribution of different Legionella species in water networks would help prevent outbreaks. Culture investigations followed by serological agglutination tests, with poly/monovalent antisera, still represent the gold standard for isolation and identification of Legionella strains. However, also MALDI-TOF and mip-gene sequencing are currently used. This study was conducted to genetically correlate strains of Legionella non pneumophila (L-np) isolated during environmental surveillance comparing different molecular techniques. Overall, 346 water samples were collected from the water system of four pavilions located in a hospital of the Apulia Region of Italy. Strains isolated from the samples were then identified by serological tests, MALDI-TOF, and mip-gene sequencing. Overall, 24.9% of water samples were positive for Legionella, among which the majority were Legionella pneumophila (Lpn) 1 (52.3%), followed by Lpn2-15 (20.9%), L-np (17.4%), Lpn1 + Lpn2-15 (7.1%), and L-np + Lpn1 (2.3%). Initially, L-np strains were identified as L. bozemanii by monovalent antiserum, while MALDI-TOF and mip-gene sequencing assigned them to L. anisa. More cold water than hot water samples were contaminated by L. anisa (p < 0.001). PFGE, RAPD, Rep-PCR, and SAU-PCR were performed to correlate L. anisa strains. Eleven out of 14 strains identified in all four pavilions showed 100% of similarity upon PFGE analysis. RAPD, Rep-PCR, and SAU-PCR showed greater discriminative power than PFGE.

Thermophilin 13: In Silico Analysis Provides New Insight in Genes Involved in Bacteriocin Production.

Bacteriocins are a large family of ribosomally synthesised proteinaceous toxins that are produced by bacteria and archaea and have antimicrobial activity against closely related species to the producer strain. Antimicrobial proteinaceous compounds are associated with a wide range of applications, including as a pathogen inhibitor in food and medical use. Among the several lactic acid bacteria (LAB) commonly used in fresh and fermented food preservation, Streptococcus thermophilus is well known for its importance as a starter culture for yoghurt and cheese. Previous studies described the bacteriocin thermophilin 13 exclusively in S. thermophilus SFi13 and the genes encoding its production as an operon consisting of two genes (thmA and thmB). However, the majority of bacteriocins possess a complex production system, which involves several genes encoding dedicated proteins with relatively specific functions. Up to now, far too little attention has been paid to the genes involved in the synthesis, regulation and expression of thermophilin 13. The aim of the present study, using in silico gene mining, was to investigate the presence of a regulation system involved in thermophilin 13 production. Results revealed the dedicated putative bacteriocin gene cluster (PBGC), which shows high similarity with the class IIb bacteriocins genes. This newly revealed PBGC, which was also found within various strains of Streptococcus thermophilus, provides a new perspective and insights into understanding the mechanisms implicated in the production of thermophilin 13.

Effect of feed restriction and refeeding on body condition, digestive functionality and intestinal microbiota in rainbow trout (Oncorhynchus mykiss).

The aim of the present work was to investigate the influence of fasting and refeeding on body condition, gut physiology and microbiota in reared O. mykiss. Ninety-six fish were randomly allotted among three groups subjected to different feeding plan: C (control, fed for 5 weeks); R (restricted ration over 3 weeks followed by 2 weeks feeding); F (fasted over 3 weeks followed by 2 weeks feeding) in a well's fresh water flow-through rearing plan. Sampling occurred at 0, 1, 2, 4, 7, 14 days during the refeeding period. At day 0 and throughout the feeding period until day 14, the weight of the fish was significantly affected by the feeding restriction. Feed deprivation reduced significantly the viscerosomatic and hepatosomatic indexes. Brush border membrane enzymes' specific activity was modulated by feeding regimes until day 7, to level in all experimental groups at day 14. At the end of the restricted/fasted period, the microbiota of the C group was made up of 70% of Actinobacteria, 24% of Proteobacteria, 4.2% of Firmicutes and < 1% of Bacteroides, while the restricted and fasted group were characterized by a strong reduction of Actinobacteria, and a significant increase in Bacteroidetes and Firmicutes. The feed deprivation determined a dysbiosis, allowing the development of different commensal or pathogenic bacteria. In conclusion, the effects of 2 weeks of feed deprivation, excluding those related to body weight, are gradually mitigated by refeeding, which allows the restoration of digestive functions and a healthy intestinal microbiota.

Lactobacilli, a Weapon to Counteract Pathogens through the Inhibition of Their Virulence Factors.

To date, several studies have reported an alarming increase in pathogen resistance to current antibiotic therapies and treatments. Therefore, the search for effective alternatives to counter their spread and the onset of infections is becoming increasingly important. In this regard, microorganisms of the former Lactobacillus genus have demonstrated the ability to reduce the virulence of pathogens. In addition to the production of bioactive substances, self- and coaggregation, and substrate competition, lactobacilli influence gene expression by downregulating genes associated with the virulence of pathogens. As demonstrated in many in vivo and in vitro trials, lactobacilli counteract and inhibit various virulence factors that favor pathogens, including the production of toxins, biofilm formation, host cell adhesion and invasion, and downregulation of virulence genes linked to quorum sensing. The aim of this review is to summarize current studies on the inhibition of pathogen virulence by lactobacilli, an important microbial group well known in the industrial and medical fields for their technological and probiotic properties that benefit human hosts with the potential to provide an important aid in the fight against pathogens besides use of the current therapies. Further research could lead to the identification of new strains that, in addition to alleviating adverse effects, could improve the efficacy of antibiotic therapies or play an important preventive role by reducing the onset of pathogen infections if regularly taken.

Microbial Characterization of Retail Cocoa Powders and Chocolate Bars of Five Brands Sold in Italian Supermarkets.

A microbial characterization of cocoa powder and chocolate bars of three batches of five different brands sold in Italian markets was performed. The results showed a variable microbial population consisting of mesophilic and thermophilic spore formation in both types of products. The chocolate bars were also contaminated with molds of environmental origin. Bacillus spp. and Geobacillus spp. were found in both products. The chocolate bars were also contaminated by molds belonging to the genera Penicillium and Cladosporium. The sporogenous strains mainly originate from the raw materials, i.e., cocoa beans, as the heat treatments involved (roasting of the beans and conching of the chocolate) are not sufficient to reach commercial sterility. Furthermore, the identified spore-forming species have often been isolated from cocoa beans. The molds isolated from chocolate seem to have an origin strictly linked to the final phases of production (environment and packaging). However, the level of contaminants is limited (<2 log CFU/g); the molds do not develop in both products due to their low Aw (<0.6) and do not affect the safety of the products. However, a case of mold development in chocolate bars was observed. Among the isolated molds, only Penicillium lanosocoeruleum demonstrated a high xero-tolerance and grew under some conditions on chocolate bars. Its growth could be explained by a cocoa butter bloom accompanied by the presence of humidity originating from the bloom or acquired during packaging.

Improving the Shelf-Life of Fish Burgers Made with a Mix of Sea Bass and Sea Bream Meat by Bioprotective Cultures.

Seafood products are one of the most perishable foods, and their shelf life is limited by enzymatic and microbial spoilage. Developing methods to extend the shelf life of fresh fish could reduce food waste in the fishery industry, retail stores, and private households. In recent decades, the application of lactic acid bacteria (LAB) as bioprotective cultures has become a promising tool. In this study, we evaluated the use of four starter cultures, previously selected for their properties as bioprotective agents, for sea bass and sea bream burgers biopreservation. Starter cultures impacted the microbial populations, biochemical parameters (pH, TVB-N), and sensory properties of fish burgers, during 10 days of storage at 4 °C and then 20 days at 8 °C in modified atmosphere packaging (MAP). Also, storage time influenced the microbial and physicochemical characteristics of all the tested samples, except for TVB-N values, which were significantly higher in the uninoculated burgers. The volatilome changed in the different treatments, and in particular, the samples supplemented with starter presented a profile that described their rapid growth and colonization, with the production of typical molecules derived from their metabolism. The addition of bioprotective cultures avoided bloating spoilage and improved the sensory parameters of the burgers. The shelf life of the fish burgers supplemented with starter cultures could be extended up to 12 days.

Effect of a triacontanol-rich biostimulant on the ripening dynamic and wine must technological parameters in Vitis vinifera cv. 'Ribolla Gialla'.

Biostimulants are organic compounds which can influence the biochemical activity of the whole plant. Lately, great attention has been focused on the possibility of using these stimulants in the viticulture sector. Due to this, the aim of this work was to investigate the foliar application of a biostimulant made by Fabaceae tissue, rich in amino acids and peptides along with the high presence of natural triacontanol (C30H62O) (>6 mg kg-1), previously reported in many crops as chemicals able to stimulate different yield components, the technological composition of musts still having an effect on some of the microbial population of different fruits/crops. Hence, this research was conducted during the growing seasons 2020 and 2021 in a commercial vineyard of the 'Ribolla Gialla' grapevine (Vitis vinifera, L.), in the Friuli Venezia Giulia Region (North-Eastern Italy), in order to understand the effect on this woody perennial crop not yet investigated. After a two-year-study, a physiological response occurred, as ripening and veraision were brought forward in the treated plants as well as the harvest time, having higher enological parameters (sugars, total titrable acidity and citric acid content) than the non-treated at every stage. Thus, grapes in the treated plants reached a full technological maturity earlier than the non-treated, in both study years. There was a positive effect on must microbial ecology important for winemaking, hence, the biostimulant have promoted the growth of the microbial community on berry skin translating into what found in the must.

Draft Genome Sequences of Eight Bacilli Isolated from an Ancient Roman Amphora.

Paleomicrobiology, the study of ancient microbiological material, allows us to understand different evolutionary phenomena in bacteria. In this study, eight bacilli isolated from an ancient Roman amphora, which dates to the IV to V sec. AD, were sequenced and functionally annotated.