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.

Zygosaccharomyces rouxii is the predominant species responsible for the spoilage of the mix base for ice cream and ethanol is the best inhibitor tested.

A mix base for ice cream (MBIC) is used to produce artisanal or industrial ice creams and desserts and consists of a mixture of different ingredients, including sugar, egg yolk, natural flavors, starch and milk proteins. MBICs, which have chemical-physical characteristics that include a pH of 5.61 and an activity water (Aw) less than or equal to 0.822, are packaged in tin boxes and stored at ambient temperature. Despite the low Aw, MBIC can support osmotolerant and osmophilic yeast growth. The aim of our work was to study the behavior of Zygosaccharomyces rouxii, the main microorganisms responsible of MBIC spoilage, either in the vivo or in a model system in order to inhibit its growth by the selection of antimicrobial agents. Different osmotolerant yeasts belonging to the genus Zygosaccharomyces were isolated and identified from spoiled and unspoiled lots of MBICs. In particular, Z. rouxii was the predominant species responsible for the spoilage, which depended on the high temperature of storage (>20 °C) and was highlighted by the presence of alcohol, esters, acids and gas (CO2), which blew open the tin boxes. To stop spoilage, different antimicrobial compounds were tested: sulfur dioxide, sorbic and benzoic acids and ethanol. However, only 2% v/v ethanol was required to achieve the total inhibition of the Z. rouxii cocktails tested in this work. The use of other antimicrobials cannot be recommended because they were not able to stop yeast spoilage and changed the color and flavor of the products. Conversely, the use of ethanol is suggested because of its extreme effectiveness against osmotolerant yeasts, and the added amount was less than or equal to the taste threshold limit. The MBICs, treated with ethanol, were stable till the end of their shelf-life (6 months).

Antibiotic resistance and virulence factors in lactobacilli: something to carefully consider.

Lactobacilli are a ubiquitous bacteria, that includes many species commonly found as part of the human microbiota, take part in the natural food fermentation processes, are used as probiotics, and in the food sector as starter cultures or bio-protectors. Their wide use is dictated by a long history of safe employ, which has allowed them to be classified as GRAS (General Recognized As Safe) microorganisms by the US Food and Drug Administration (FDA) and QPS (Qualified Presumption of Safety) by the European Food Safety Authority (EFSA, 2007; EFSA, 2021). Despite their classification as safe microorganisms, several studies show that some members of Lactobacillus genus can cause, especially in individuals with previous pathological conditions, problems such as bacteremia, endocarditis, and peritonitis. In other cases, the presence of virulence genes and antibiotic resistance, and its potential transfer to pathogenic microorganisms constitute a risk to be considered. Consequently, their safety status was sometimes questioned, and it is, therefore, essential to carry out appropriate assessments before their use for any purposes. The following review focuses on the state of the art of studies on genes that confer virulence factors, including antibiotic resistance, reported in the literature within the lactobacilli, defining their genetic basis and related functions.

A survey of a blown pack spoilage produced by Clostridium perfringens in vacuum-packaged wurstel.

Three hundred Clostridium strains were isolated from spoiled wurstels and were identified by traditional and molecular methods as Clostridium perfringens. The phenotypic characteristics of the strains were studied. All the strains produced acetic and butyric acids and enterotoxin. C. perfringens grew in the spoiled wurstels because it was present in raw meat (Lot 150) at a level of 3.2 log CFU/g due to an unchecked cooling phase that took 28 h to decrease the temperature of the wurstels from 60 to 9-10 °C, which is the lower limit for C. perfringens growth. During the 28 h of cooling, the concentration of C. perfringens increased to 6.5 CFU/g. It was concluded that its presence and the long cooling time were the main factors responsible for the spoilage. Wurstels intentionally made with contaminated meat (3 log CFU/g) but cooled after cooking for 17 h to 9 °C did not support C. perfringens growth; consequently, these wurstels remained unspoiled. The packages of the spoiled wurstels were blown, and the products were soft (soggy), textureless and had the odour of acetic acid, ethanol and sulfur.

Microbial quality of raw and ready-to-eat mung bean sprouts produced in Italy.

The aim of this study was to determine the microbial quality of mung bean sprouts produced in Italy. The presence of pathogenic microorganisms (Shiga toxin-producing Escherichia coli (STEC), Salmonella spp. and Listeria monocytogenes), total coliforms, and total viable counts (TVCs) were determined. The study covered five years of sprout production. The results demonstrated that no pathogenic microorganisms were present, and the microbial load was less than 6 log CFU/g. The mung bean sprouts currently produced in Italy were found to be acceptable for consumption. An additional aim was to determine the fate of different strains of STEC, L. monocytogenes and Salmonella spp. by intentionally inoculating mung bean seeds during sprouting and by using chlorinated water to reduce the concentration of these strains in seeds and sprouts. The data demonstrated that these strains increased over 5-6 log CFU/g within 3 days from inocula. The chlorinated washing solution reduced the concentration of the investigated strains in seeds and sprouts by approximately 3 and 7 log CFU/g, respectively. However, it was not possible to completely eliminate the pathogens from either the mung bean seeds or sprouts. Despite these encouraging results, the producer's attention to hygienic quality should not be reduced when attempting to produce safe-to-consume mung bean sprouts.

Fate of the microbial population and the physico-chemical parameters of "Sanganel" a typical blood sausages of the Friuli, a north-east region of Italy.

In Friuli, a Northeastern region of Italy, a blood sausage called Sanganel is produced by farmers, butchers, shops, and factories. This sausage is made with pork meat, boiled blood, lard, spices, and salt. It is stored at 4 ± 2 °C and usually eaten fresh or boiled within 14 days of its manufacture. Little is known about its microbial populations and safety for consumption. The aim of this study is to characterise the microbial populations and the physico-chemical parameters of Sanganel to establish its quality and the safety of consuming it. The microbial population of Sanganel is typical of meat products, and psychrotrophic enterobacteria and lactic acid bacteria (LAB) grow while it is stored. Enterobacteria produce total basic volatile nitrogen (TVB-N) and biogenic amines that, despite the presence of LAB, increase the pH of the sausage to approximately 6.9. Considering the concentrations of Enterobacteriaceae and TVB-N in the sausage, a shelf-life of 14 days is suggested. However, at 30 days the sausage is safe to eat and presents normal odours and flavours. In addition, boiling the sausage for 30 min before consumption eliminates the asporogenous microbial population.

Biocontrol of ochratoxigenic moulds (Aspergillus ochraceus and Penicillium nordicum) by Debaryomyces hansenii and Saccharomycopsis fibuligera during speck production.

Speck is a meat product obtained from the deboned leg of pork that is salted, smoked and seasoned for four to six months. During speck seasoning, Eurotium rubrum and Penicillium solitum grow on the surface and collaborate with other moulds and tissue enzymes to produce the typical aroma. Both of these strains usually predominate over other moulds. However, moulds producing ochratoxins, such as Aspergillus ochraceus and Penicillium nordicum, can also co-grow on speck and produce ochratoxin A (OTA). Consequently, speck could represent a potential health risk for consumers. Because A. ochraceus and P. nordicum could represent a problem for artisanal speck production, the aim of this study was to inhibit these mould strains using Debaryomyces hansenii and Saccharomycopsis fibuligera. Six D. hansenii and six S. fibuligera strains were tested in vitro to inhibit A. ochraceus and P. nordicum. The D. hansenii DIAL 1 and S. fibuligera DIAL 3 strains demonstrated the highest inhibitory activity and were selected for in vivo tests. The strains were co-inoculated on fresh meat cuts for speck production with both of the OTA-producing moulds prior to drying and seasoning. At the end of seasoning (six months), OTA was not detected in the speck treated with both yeast strains. Because the yeasts did not adversely affect the speck odour or flavour, the strains are proposed as starters for the inhibition of ochratoxigenic moulds.

Lactococcus lactis and Lactobacillus sakei as bio-protective culture to eliminate Leuconostoc mesenteroides spoilage and improve the shelf life and sensorial characteristics of commercial cooked bacon.

Cooked bacon is a typical Italian meat product. After production, cooked bacon is stored at 4 ± 2 °C. During storage, the microorganisms that survived pasteurisation can grow and produce spoilage. For the first time, we studied the cause of the deterioration in spoiled cooked bacon compared to unspoiled samples. Moreover, the use of bio-protective cultures to improve the quality of the product and eliminate the risk of spoilage was tested. The results show that Leuconostoc mesenteroides is responsible for spoilage and produces a greening colour of the meat, slime and various compounds that result from the fermentation of sugars and the degradation of nitrogen compounds. Finally, Lactococcus lactis spp. lactis and Lactobacillus sakei were able to reduce the risk of Leuconostoc mesenteroides spoilage.

A new cause of spoilage in goose sausages.

The aim of this work was to determine the microorganisms present and to investigate their metabolites that cause spoilage of many goose sausages produced in Friuli, a northeast region of Italy. The defect was observed by sensorial analysis using the "needle probing" technique; the spoiled sausages were unsafe and not marketable. Despite the addition of starter, the microorganisms, particularly enterococci and Enterobacteriaceae, grew during ripening and produced a large amount of biogenic amines; therefore, these sausages represented a risk to consumers. The production of those compounds was confirmed in vitro. Furthermore, a second cause of spoilage was attributed to moulds that grew during ripening; the fungi grew between the meat and casing, producing a large amount of total volatile nitrogen, and consequently an ammonia smell was present either in the ripening area or in the sausages. This is the first description of this type of defect in goose sausages.

High resolution melting analysis (HRM) as a new tool for the identification of species belonging to the Lactobacillus casei group and comparison with species-specific PCRs and multiplex PCR.

The correct identification and characterisation of bacteria is essential for several reasons: the classification of lactic acid bacteria (LAB) has changed significantly over the years, and it is important to distinguish and define them correctly, according to the current nomenclature, avoiding problems in the interpretation of literature, as well as mislabelling when probiotic are used in food products. In this study, species-specific PCR and HRM (high-resolution melting) analysis were developed to identify strains belonging to the Lactobacillus casei group and to classify them into L. casei, Lactobacillus paracasei and Lactobacillus rhamnosus. HRM analysis confirmed to be a potent, simple, fast and economic tool for microbial identification. In particular, 201 strains, collected from International collections and attributed to the L. casei group, were examined using these techniques and the results were compared with consolidated molecular methods, already published. Seven of the tested strains don't belong to the L. casei group. Among the remaining 194 strains, 6 showed inconsistent results, leaving identification undetermined. All the applied techniques were congruent for the identification of the vast majority of the tested strains (188). Notably, for 46 of the strains, the identification differed from the previous attribution.

Use of propidium monoazide for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR.

Brettanomyces bruxellensis is a current problem in winemaking all over the world, and the question if B. bruxellensis has a positive or negative impact on wine is one of the most controversial discussions in the world. The presence of live B. bruxellensis cells represents the risk of growth and an increase in cell numbers, which is related to the potential production of volatile phenols. In this work, the optimisation of a PMA-quantitative PCR (qPCR) method to enumerate only viable cells was carried out using the standard strain B. bruxellensis DSMZ 70726. The obtained detection limits were 0.83 log CFU/mL in red wine, 0.63 log CFU/mL in white wine and 0.23 log CFU/mL in beer. Moreover, the quantification was also performed by Reverse Transcription quantitative PCR (RT-qPCR), and the results showed a higher detection limit for all of the trials.

Rapid detection and differentiation of important Campylobacter spp. in poultry samples by dot blot and PCR.

The detection of Campylobacter, the most commonly reported cause of foodborne gastroenteritis in the European Union, is very important for human health. The most commonly recognised risk factor for infection is the handling and/or consumption of undercooked poultry meat. The methods typically applied to evaluate the presence/absence of Campylobacter in food samples are direct plating and/or enrichment culture based on the Horizontal Method for Detection and Enumeration of Campylobacter spp. (ISO 10272-1B: 2006) and PCR. Molecular methods also allow for the detection of cells that are viable but cannot be cultivated on agar media and that decrease the time required for species identification. The current study proposes the use of two molecular methods for species identification: dot blot and PCR. The dot blot method had a sensitivity of 25 ng for detection of DNA extracted from a pure culture using a digoxigenin-labelled probe for hybridisation; the target DNA was extracted from the enrichment broth at 24 h. PCR was performed using a pair of sensitive and specific primers for the detection of Campylobacter jejuni and Campylobacter coli after 24 h of enrichment in Preston broth. The initial samples were contaminated by 5 × 10 C. jejuni cells/g and 1.5 × 10(2)C. coli cells/g, thus the number of cells present in the enrichment broth at 0 h was 1 or 3 cell/g, respectively.

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.

Use of propidium monoazide for the enumeration of viable Oenococcus oeni in must and wine by quantitative PCR.

Malolactic fermentation is an important step in winemaking, but it has to be avoided in some cases. It's carried out by lactic acid bacteria belonging mainly to the genus Oenococcus, which is known to be a slow growing bacterium. Classical microbiological methods to enumerate viable cells of Oenococcus oeni in must and wine take 7-9 days to give results. Moreover, RT-qPCR technique gives accurate quantitative results, but it requires time consuming steps of RNA extraction and reverse transcription. In the present work we developed a fast and reliable quantitative PCR (qPCR) method to enumerate cells of Oenococcus oeni, directly, in must and wine. For the first time we used a propidium monoazide treatment of samples to enumerate only Oenococcus oeni viable cells. The detection limit of the developed method is 0.33 log CFU/mL (2.14 CFU/mL) in must, and 0.69 log CFU/mL (4.90 CFU/mL) in wine, lower than that of the previously developed qPCR protocols.

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.

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.

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.

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.

Catalase-positive cocci in fermented sausage: Variability due to different pork breeds, breeding systems and sausage production technology.

The aim of this study was to compare the ecology of catalase-positive cocci (CPC) present in traditional fermented sausages produced using different breeds of pork, each of which was raised in two different environments and processed using two different technologies. Semi-quantitative molecular methods were used to determine bacterial identities. Almost all fermentations were characterised by a significant increase in CPC during the first few days of fermentation, reaching values of 10(5)-10(6) cfu g(-1) within 3 days. Staphylococcus xylosus and Staphylococcus equorum species, which were detected over the course of fermentation, were found to be the predominant population in all the monitored fermentation. Staphylococcus haemolyticus, Staphylococcus lentus, Micrococcus luteus, Macrococcus caseolyticus and Staphylococcus succinus were also present, but their concentrations were found to vary under the different experimental conditions. Using cluster analysis, we concluded that a plant-specific CPC ecology existed. In addition, the breed of pork used for production was found to influence the presence of some CPC species. However, from this study, it was not possible to reach the same conclusion regarding the breeding system used.