Dual effects of a bacteriocin-producing Lactiplantibacillus pentosus CF-6HA, isolated from fermented aloreña table olives, as potential probiotic and antimicrobial agent.

The probiotic potential of Lactiplantibacillus pentosus CF-6HA isolated from traditionally fermented Aloreña table olives was analyzed in vitro and in silico. Results obtained suggested that this strain can be catalogued as "talented" bacterium exhibiting bacteriocin production with antimicrobial activity against human/animal and plant pathogens, such as Pseudomonas syringae and Verticillium dahliae. The robustness, safety and probiotic potential of L. pentosus CF-6HA was confirmed by in silico analysis. In addition, a plethora of coding genes for defense and adaptability to different life styles besides functional properties were identified. In this sense, defense mechanisms of L. pentosus CF-6HA consist of 17 ISI elements, 98 transposases and 13 temperate phage regions as well as a CRISPR (clustered regularly interspaced short palindromic repeats)/cas system. Moreover, the functionality of this strain was confirmed by the presence of genes coding for secondary metabolites, exopolysaccharides and other bioactive molecules. Finally, we demonstrated the ability of L. pentosus CF-6HA to biotransform selenite to nanoparticles (SeNPs) highlighting its potential role in selenium bioremediation to be exploited in foods, agriculture and the environment; but also for the bio-enrichment of fermented foods with selenium.

Antimicrobial profile of the culturable olive sporobiota and its potential as a source of biocontrol agents for major phytopathogens in olive agriculture.

BACKGROUND: The phytopathogens Xylella fastidiosa and Verticillium dahliae present an unparalleled threat to olive agriculture. However, there is no efficient field treatment available today for either pest. Spore-forming bacteria (i.e., the sporobiota) are known for their extraordinary resistance properties and antimicrobial activity. The aim of this study was thus to identify potential novel sustainable spore-forming biocontrol agents derived from the culturable olive microbiome, termed the sporobiota, in general and in particular against X. fastidiosa and V. dahliae. RESULTS: We demonstrate the wide-ranging antimicrobial profile of 415 isolates from the culturable olive sporobiota towards human and plant pathogens. We further identified five candidates with antagonistic activity against X. fastidiosa and V. dahliae. These belong to the Bacillus subtilis, Bacillus cereus and Peribacillus simplex clade. The activity was related to the species and their relative origin (soil versus leaf endophytic). It is of particular interest that two of these candidates are already naturally present at the site of disease-development that is, plant interior. We further confirmed the presence of lipopeptide genes potentially associated with the reported bioactivity. CONCLUSIONS: The study provides insights into how members of the olive sporobiota may support the olive plant to ward off detrimental pathogens. It further yields five promising candidates for the development of eco-friendly, multi-active biocontrol agents in olive agriculture. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Simply Versatile: The Use of Peribacillus simplex in Sustainable Agriculture.

Peribacillus simplex is a Gram-positive, spore-forming bacterium derived from a vast range of different origins. Notably, it is part of the plant-growth-promoting rhizobacterial community of many crops. Although members of the Bacillaceae family have been widely used in agriculture, P. simplex has, so far, remained in the shadow of its more famous relatives, e.g., Bacillus subtilis or Bacillus thuringiensis. Recent studies have, however, started to uncover the bacterium's highly promising and versatile properties, in particular in agricultural and environmental applications. Hence, here, we review the plant-growth-promoting features of P. simplex, as well as its biocontrol activity against a variety of detrimental plant pests in different crops. We further highlight the bacterium's potential as a bioremediation agent for environmental contaminants, such as metals, pesticide residues, or (crude) oil. Finally, we examine the recent developments in the European regulatory landscape to facilitate the use of microorganisms in plant protection products. Undoubtedly, further studies on P. simplex will reveal additional benefits for agricultural and environmentally friendly applications.

Metagenomic insights into microbial contamination in critical healthcare environments and the efficacy of a novel "HLE" disinfectant.

BACKGROUND: Bacterial contamination on inanimate clinical surfaces is directly linked to severe health problems, especially those caused by multidrug resistant (MDR) pathogens. Here, we evaluated the microbial burden in these environments and tested the efficacy of a novel HLE disinfectant solution. METHODS: Microbial contamination of healthcare surfaces [Intensive Care Unit (ICU), Long Period Hospitalization Room (LPHR) and Otolaryngology Consultation (OC)] and the efficacy of HLE disinfectant solution were determined analyzing the viable counts on general and selective media, and also by molecular studies focused on metagenomic and specific qPCR. RESULTS: Different contamination loads were detected with LPHR showing the highest contamination. Treatment with the HLE disinfectant solution curbed the spread of well-adapted pathogens on touched surfaces (ICU, LPHR and OC). Metagenomic analysis of microbial diversity of the Patient Table (most contaminated surface in LPHR) revealed the presence of mainly A. johnsonii and P. putida. Furthermore, functional annotation of toxin, virulence and antibiotic resistance sequences showed a high diversity of Acinetobacter spp. and Pseudomonas spp. In this context, specific qPCR analysis confirmed the efficacy of HLE disinfectant solution against the most prevalent and critical pathogens Pseudomonas sp. and Acinetobacter sp. achieving their complete eradication. CONCLUSION: Given the persistence of detrimental resistant pathogens, the application of HLE disinfection solution could be a highly beneficial and effective option -used either alone or in combination-for infection prevention and control with the aim to eliminate microbial pathogens and their genes from contaminated contact-surfaces and thus limit the spread to humans and other ecological niches.

Corrigendum: In silico genomic analysis of the potential probiotic Lactiplantibacillus pentosus CF2-10N reveals promising beneficial effects with health promoting properties.

[This corrects the article DOI: 10.3389/fmicb.2022.989824.].

Novel combination of nanoparticles and metallo-ß-lactamase inhibitor/antimicrobial-based formulation to combat antibiotic resistant Enterococcus sp. and Pseudomonas sp. strains.

Nanotechnology presents an innovative strategy to combat the spread of antibiotic resistant bacteria and their resistance genes throughout different ecosystems. To address this challenge, nanoparticles (silver, gold, zinc and copper) alone or in combination with metallo-ß-lactamase inhibitor/antimicrobial-based formulation (EDTA/HLE) showed antimicrobial activity against antibiotic resistant Enterococcus sp. and Pseudomonas sp. strains. Furthermore, the observed synergistic effect was detected notably for silver, zinc or copper nanoparticles with EDTA (ethylenediaminetetraacetic acid) and silver nanoparticles with HLE against planktonic Enterococcus sp. strains, or gold nanoparticles+EDTA or HLE against Pseudomonas sp. Regarding activity against bacterial biofilms, zinc nanoparticles combined with either of the reagents caused strong inhibition of developing biofilms of antibiotic resistant Enterococcus sp. Pseudomonas sp. strains, while preformed biofilms were mainly inhibited by silver nanoparticles+reagent. Microscopic analyses confirmed that the antimicrobial activity of nanoparticles was caused by adsorption to the bacterial cell surface, and further enhanced by chelating agents. Hence, we can conclude that nanoparticles+EDTA or HLE could represent a good alternative to limit the spread of antibiotic resistant bacteria in the food chain and the environment.

Special Issue "Beneficial and Detrimental Microorganisms Occurring in Fermented Foods": Editorial.

Numerous and heterogeneous populations of beneficial microorganisms originating from raw materials, equipment, and production and processing environments can affect the fermentation process by their metabolic activities, allowing for the enhancement of the nutritional value, sensory characteristics, overall quality, safety, and shelf-life of final food products [...].

Characterization of the Culturable Sporobiota of Spanish Olive Groves and Its Tolerance toward Environmental Challenges.

Olive agriculture presents an integral economic and social pillar of the Mediterranean region with 95% of the world's olive tree population concentrated in this area. A diverse ecosystem consisting of fungi, archaea, viruses, protozoa, and microbial communities-the soil microbiome-plays a central role in maintaining healthy soils while keeping up productivity. Spore-forming organisms (i.e., the sporobiota) have been identified as one of the predominant communities of the soil microbiome and are known for the wide variety of antimicrobial properties and extraordinary resistance. Hence, the aim of this work was to determine the culturable sporobiota of Spanish olive orchards and characterize its phenotypic properties toward common environmental challenges. A collection of 417 heat-resistant bacteria were isolated from five Spanish olive orchards. This collective was termed the "olive sporobiota." Rep-PCR clustering of representative isolates revealed that they all belonged to the group of Bacillus spp., or closely related species, showing a great variety of species and strains. Representative isolates showed susceptibility to common antibiotics, as well as good resistance to heavy metal exposure, with an order of metal tolerance determined as iron > copper > nickel > manganese > zinc > cadmium. Finally, we showed that the application of mineral fertilizer can in several cases enhance bacterial growth and thus potentially increase the relative proportion of the sporobiota in the olive grove ecosystem. In summary, the identification of the culturable olive sporobiota increases our understanding of the microbial diversity in Spanish olive groves, while tolerance and resistance profiles provide important insights into the phenotypic characteristics of the microbial community. IMPORTANCE Microbial communities are a key component of healthy soils. Spore-forming microorganisms represent a large fraction of this community-termed the "sporobiota"-and play a central role in creating a conducive environment for plant growth and food production. In addition, given their unique features, such as extraordinary stability and antimicrobial properties, members of the sporobiota present interesting candidates for biotechnological applications, such as sustainable plant protection products or in a clinical setting. For this, however, more information is needed on the spore-forming community of agricultural installations, ultimately promoting a transition toward a more sustainable agriculture.

Changes in resistome profile of potential probiotic Lactiplantibacillus pentosus in response to edible oil adaptation.

Despite increasing interest to investigate horizontal gene transfer as a leading cause of antibiotic resistance spread, the resistome is not only influenced by the influx and efflux of genes in different environments. Rather, the expression of existing genes under different stress conditions requires special attention. This study determined whether pre-adapting Lactiplantibacillus pentosus strains, isolated from Aloreña green table olives, to vegetable-based edible oils influence their phenotypic and genotypic responses to antibiotics. This has significant diet, food matrix, gut health, and food safety concerns. Pre-adapting L. pentosus strains to oils significantly changed their susceptibility profile to antibiotics. However, results generally differed among the three strains; although changes in the Minimum Inhibitory Concentration (MIC) of antibiotics occurred, it depended on the L. pentosus strain and the oil used for adaptation. The pre-adaptation of L. pentosus strains with olive, sunflower, argan and linseed oils induced gene expressions (e.g., rpsL, recA and uvrB) in several stress responses. Thus, to analyze this fact in-depth, transcriptional changes were reported in the selected potential probiotic L. pentosus CF2-10 adapted with olive or sunflower, rerouting its metabolic pathways to export toxic molecules through efflux pumps and ABC transporters. Pre-adaptation of some lactobacilli with olive or sunflower oils may represent a novel approach for manufacturing probiotic products with improved stability, functionality and robustness.

In silico genomic analysis of the potential probiotic Lactiplantibacillus pentosus CF2-10N reveals promising beneficial effects with health promoting properties.

Lactiplantibacillus pentosus CF2-10 N, isolated from brines of naturally fermented Aloreña green table olives, exhibited high probiotic potential. High throughput sequencing and annotation of genome sequences underline the potential of L. pentosus CF2-10 N as excellent probiotic candidate of vegetable origin. In a previous study we could show the probiotic potential of CF2-10 N in vitro, while in this study in silico analysis of its genome revealed new insights into its safety and functionality. Our findings highlight the microorganism's ecological flexibility and adaptability to a broad range of environmental niches, food matrices and the gastrointestinal tract. These features are shared by both phylogenetically very close L. pentosus strains (CF2-10 N and MP-10) isolated from the same ecological niche with respect to their genome size (≅ 3.6 Mbp), the presence of plasmids (4-5) and several other properties. Nonetheless, additional and unique features are reported in the present study for L. pentosus CF2-10 N. Notably, the safety of L. pentosus CF2-10 N was shown by the absence of virulence determinants and the determination of acquired antibiotic resistance genes, i.e., resistome, which is mostly represented by efflux-pump resistance genes responsible for the intrinsic resistance. On the other hand, defense mechanisms of L. pentosus CF2-10 N include eight prophage regions and a CRISPR/cas system (CRISPR-I and CRISPR-II) as acquired immune system against mobile elements. Finally, the probiotic potential of this strain was further demonstrated by the presence of genes coding for proteins involved in adhesion, exopolysaccharide biosynthesis, tolerance to low pH and bile salts, immunomodulation, and vitamin and enzyme production. Taken together these results, we propose the use of L. pentosus CF2-10 N as a potential and promising probiotic candidate able to colonize several niches and adapt to different lifestyles. The strain can provide attractive functional and probiotic features necessary for its application as starter culture and probiotic.

Antibacterial and antibiofilm effects of essential oil components, EDTA and HLE disinfectant solution on Enterococcus, Pseudomonas and Staphylococcus sp. multiresistant strains isolated along the meat production chain.

The spread of multidrug resistant (MDR) bacteria and resistance genes along the food chain and the environment has become a global, but silent pandemic. To face this challenge, it is of outmost importance to develop efficient strategies to reduce potential contamination by these agents. In the present study, 30 strains of Enterococcus sp., Staphylococcus sp. and Pseudomonas sp. isolated from various surfaces throughout the meat production chain in a goat and lamb slaughterhouse were characterized as MDR bacteria harboring several antibiotic resistance genes (ARGs). The antimicrobial efficacy of natural essential oil components "EOCs" (carvacrol "CA," cinnamaldehyde "CIN," eugenol "EU," geraniol "GE," limonene "LI" and thymol "TH"), HLE disinfectant solution (3-6% H2O2; 2.2-4.4% lactic acid and 12.5-25 mM EDTA in water) and EDTA was tested against these MDR bacteria. Results showed that Minimum Inhibitory Concentrations (MIC) were compound and strain dependent. In addition, the synergistic effect of these antimicrobials was evaluated at 1/2 MIC. Here our study showed particularly promising results regarding the inhibitory effect at sub-inhibitory concentrations, which were confirmed by the analysis of bacterial growth dynamics over 72 h. Furthermore, the inhibitory effect of EOCs, HLE disinfectant solution and EDTA or their combinations was studied in developing and established biofilms of MDR bacteria obtaining variable results depending on the morphological structure of the tested strain and the phenolic character of the EOCs. Importantly, the combination of EOCs with HLE or EDTA showed particularly positive results given the effective inhibition of biofilm formation. Moreover, the synergistic combinations of EU and HLE/EDTA, TH, CA, GE, LI or CIN + EDTA/HLE caused log reductions in established biofilms of several strains (1-6 log10 CFU) depending on the species and the combination used, with Pseudomonas sp. strains being the most susceptible. Given these results, we propose novel antimicrobial formulations based on the combination of sub-inhibitory concentrations of EOCs and HLE or EDTA as a highly promising alternative to currently used approaches. This novel strategy notably shows great potential to efficiently decrease the emergence and spread of MDR bacteria and ARGs in the food chain and the environment, thus supporting the decrease of resistomes and pathogenesis in clinical and industrial areas while preserving the antibiotic therapeutic action.

Transcriptomic Profile and Probiotic Properties of Lactiplantibacillus pentosus Pre-adapted to Edible Oils.

In this study, we determined whether pre-adapting Lactiplantibacillus pentosus strains, isolated from Aloreña green table olives, to vegetable-based edible oils improved their robustness and functionality; this may have great importance on their stress response during fermentation, storage, and digestion. Pre-adapting the strains to the corresponding oils significantly increased their probiotic functionality (e.g., auto-aggregation, co-aggregation with pathogens, and mucin adhesion), although results depended on the strain and the oil used for pre-adaptation. As such, we selected olive-adapted (TO) L. pentosus AP2-16, which exhibited improved functionality, and subjected it to transcriptomic profiling with the aim to understand the molecular mechanisms involved in the adaptation and the increased functionality. Global transcriptomic analysis of oil-adapted (olive or almond) and non-adapted (control) L. pentosus AP2-16 realized that 3,259 genes were expressed, with 2,779 mapped to the reference database. Comparative transcriptomic analysis showed that 125 genes (olive vs. control) and 108 genes (olive vs. almond) became significantly differentially expressed. TO L. pentosus AP2-16 responded by rerouting its metabolic pathways to balance energy production and storage, cell growth and survivability, host interactions (glycoconjugates), and other physiological features. As such, the pre-adaptation of lactobacilli with olive oil switches their transcriptional network to regulate robustness and functionality, possibly representing a novel approach toward the design and manufacture of probiotic products with improved stability and functionality.

Molecular identification and antibiotic resistance of bacteriocinogenic lactic acid bacteria isolated from table olives.

In the present study, lactic acid bacteria were isolated from table olive in Morocco. Random Amplified Polymorphic DNA fingerprinting with (GTG)'(5) primer revealed a remarquable variability within isolates. According to the molecular identification, Enterococcus faecium was the most dominant species isolated with 32 strains (84.21%), followed by 4 strains of Weissella paramesenteroides (10.52%), 1 strain of Leuconostoc mesenteroides (2.63%) and Lactobacillus plantarum (2.63%). All of the strains that were identified showed occurrence of more than one bacteriocin-encoding gene. Based on the results obtained, L. plantarum 11 showed a mosaic of loci coding for nine bacteriocins (pln A, pln D, pln K, pln G, pln B, pln C, pln N, pln J, ent P). A phenotypic and genotypic antibiotic resistance was also examined. L. plantarum 11, L. mesenteroides 62, W. paramesenteroides 9 and W. paramesenteroides 36 as well as all the strains of E. faecium were susceptible to ampicillin, clindamycin and teicoplanin; however, isolates showed a resistance profile against tetracycline and erythromycin. Except E. faecium 114, E. faecium 130 and L. plantarum 11, no antibiotic resistance genes were detected in all of the strains, which might be due to resistances resulting from non-transferable or non-acquired resistance determinants (intrinsic mechanism).

In silico mapping of microbial communities and stress responses in a porcine slaughterhouse and pork products through its production chain, and the efficacy of HLE disinfectant.

The use of shotgun metagenomic sequencing to understand ecological-level spread of microbes and their genes has provided new insights for the prevention, surveillance and control of microbial contaminants in the slaughterhouse environment. Here, microbial samples were collected from products and surrounding areas though a porcine slaughter process; shotgun metagenomic DNA-sequencing of these samples revealed a high community diversity within the porcine slaughterhouse and pork products, in zones originating from animal arrival through to the sale zones. Bacteria were more prevalent in the first zones, such as arrival- and anesthesia-zones, and DNA viruses were prevalent in the scorching-and-whip zone, animal products and sale zone. Data revealed the dominance of Firmicutes and Proteobacteria phyla followed by Actinobacteria, with a clear shift in the relative abundance of lactic acid bacteria (mainly Lactobacillus sp.) from early slaughtering steps to Proteobacteria and then to viruses suggesting site-specific community compositions occur in the slaughterhouse. Porcine-type-C oncovirus was the main virus found in slaughterhouse, which causes malignant diseases in animals and humans. As such, to guarantee food safety in a slaughterhouse, a better decipher of ecology and adaptation strategies of microbes becomes crucial. Analysis of functional genes further revealed high abundance of diverse genes associated with stress, especially in early zones (animal and environmental surfaces of arrival zone with 57,710 and 40,806 genes, respectively); SOS responsive genes represented the most prevalent, possibly associated with genomic changes responsible of biofilm formation, stringent response, heat shock, antimicrobial production and antibiotic response. The presence of several antibiotic resistance genes suggests horizontal gene transfer, thus increasing the likelihood for resistance selection in human pathogens. These findings are of great concern, with the suggestion to focus control measures and establish good disinfection strategies to avoid gene spread and microbial contaminants (bacteria and viruses) from the animal surface into the food chain and environment, which was achieved by applying HLE disinfectant after washing with detergent.

The life and times of yeasts in traditional food fermentations.

Yeasts are eukaryotic microorganisms which have a long history in the biotechnology of food production, as they have been used since centuries in bread-making or in the production of alcoholic beverages such as wines or beers. Relative to this importance, a lot of research has been devoted to the study of yeasts involved in making these important products. The role of yeasts in other fermentations in association with other microorganisms - mainly lactic acid bacteria - has been relatively less studied, and often it is not clear if yeasts occurring in such fermentations are contaminants with no role in the fermentation, spoilage microorganisms or whether they actually serve a technological or functional purpose. Some knowledge is available for yeasts used as starter cultures in fermented raw sausages or in the production of acid curd cheeses. This review aimed to summarize the current knowledge on the taxonomy, the presence and potential functional or technological roles of yeasts in traditional fermented plant, dairy, fish and meat fermentations.

New insights into the role of plasmids from probiotic Lactobacillus pentosus MP-10 in Aloreña table olive brine fermentation.

In silico analysis of Lactobacillus pentosus MP-10 plasmids (pLPE-1 to pLPE-5) suggests that plasmid-borne genes mediate the persistence of lactobacilli during olive fermentation and enhance their probiotic properties and their competitiveness in several ecological niches. The role of plasmids in the probiotic activities of L. pentosus MP-10 was investigated by plasmid-curing process which showed that plasmids contribute in increased metal tolerance and the biosequestration of several metals such as iron, aluminium, cobalt, copper, zinc, cadmium and mercury. Statistically significant differences in mucin adhesion were detected between the uncured and the cured L. pentosus MP-10, which possibly relied on a serine-rich adhesin (sraP) gene detected on the pLPE-2 plasmid. However, plasmid curing did not affect their tolerance to gastro-intestinal conditions, neither their growth ability under pre-determined conditions, nor auto-aggregation and pathogen co-aggregation were changed among the cured and uncured L. pentosus MP-10. These findings suggest that L. pentosus MP-10 plasmids play an important role in gastro-intestinal protection due to their attachment to mucin and, thus, preventing several diseases. Furthermore, L. pentosus MP-10 could be used as a bioquencher of metals in the gut, reducing the amount of these potentially toxic elements in humans and animals, food matrices, and environmental bioremediation.

Deciphering Resistome and Virulome Diversity in a Porcine Slaughterhouse and Pork Products Through Its Production Chain.

We aimed to better understand resistome and virulome patterns on animal and process-area surfaces through a pig slaughterhouse to track possible contamination within the food production chain. Culture-dependent methods revealed high levels of microbial contamination, corresponding to mesophilic and pathogenic bacteria on both the animal and process-area surfaces mainly in the anesthesia (AA and AS) zone followed by "scorching and whip" (FA and FS) zone and also in the end products. To evaluate the potential risk of antibiotic resistance and virulence determinants, shotgun metagenomic DNA-sequencing of isolates from selected areas/products uncovered a high diversity and richness of antibiotic resistance genes (ARGs): 55-62 genes in the anesthesia area (AA and AS) and 35-40 in "animal-arrival zone" (MA and MS). The "scorching and whip" (FA and FS) area, however, exhibited lowered abundance of ARGs (1-6), indicating that the scalding and depilating process (an intermediate zone between "anesthesia" and "scorching and whip") significantly decreased bacterial load by 1-3 log10 but also diminished the resistome. The high prevalence of antibiotic-inactivating enzyme genes in the "animal-arrival zone" (60-65%) and "anesthesia" area (56%) were mainly represented by those for aminoglycoside (46-51%) and lincosamide (14-19%) resistance, which did not reflect selective pressures by antibiotics most commonly used in pig therapy-tetracyclines and beta-lactams. Contrary to ARGs, greater number of virulence resistance genes were detected after evisceration in some products such as kidney, which reflected the poor hygienic practices. More than 19 general virulence features-mainly adherence, secretion system, chemotaxis and motility, invasion and motility were detected in some products. However, immune evasion determinants were detected in almost all samples analyzed from the beginning of the process, with highest amounts found from the anesthesia area. We conclude that there are two main sources of contamination in a pig slaughterhouse: the microorganisms carried on the animals' hide, and those from the evisceration step. As such, focussing control measures, e.g., enhanced disinfection procedures, on these contamination-source areas may reduce risks to food safety and consumer health, since the antibiotic and virulence determinants may spread to end products and the environment; further, ARG and virulence traits can exacerbate pathogen treatments.

Proteomic analysis of Lactobacillus pentosus for the identification of potential markers of adhesion and other probiotic features.

We analyzed the adhesion capacity to mucus of 31 Lactobacillus pentosus strains isolated from naturally fermented Aloreña green table olives using an immobilized mucin model. On the basis of their adhesive capacity to mucin, three phenotypes were selected for cell-wall protein proteomic analysis to pinpoint proteins involved in the adhesion process: the highly adhesive L. pentosus CF1-43 N (73.49% of adhesion ability), the moderately adhesive L. pentosus CF1-37 N (49.56% of adhesion ability) and the poorly adhesive L. pentosus CF2-20P (32.79% of adhesion ability). The results revealed four moonlighting proteins over-produced in the highly adhesive L. pentosus CF1-43 N, which were under/not produced in the other two L. pentosus strains (CF1-37 N and CF2-20P). These proteins were involved in glycolytic pathway (phosphoglycerate mutase and glucosamine-6-phosphate deaminase), stress response (small heat shock protein) and transcription (transcription elongation factor GreA). Furthermore, the relative fold change in gene expression analysis showed significant up-regulation of the genes coding for these four moonlighting proteins in the highly adhesive L. pentosus CF1-43 N versus the poorly adhesive L. pentosus CF2-20P and also in response to mucin for 20 h which clearly indicate the significant role of these genes in the adhesion capacity of L. pentosus. Thus, these proteins could be used as biomarkers for mucus adhesion in L. pentosus. On the other hand, mucin exposure induced other probiotic effects in L. pentosus strains, enhancing their co-aggregation ability with pathogens and possible inactivation.

Efficacy of "HLE"-a multidrug efflux-pump inhibitor-as a disinfectant against surface bacteria.

We evaluated the efficacy of a new disinfectant product, HLE, to inhibit multiple species of planktonic and biofilm bacterial cultures. The HLE disinfectant comprised of EDTA, lactic acid and hydrogen peroxide, and our data indicated that the disinfectant had effective antimicrobial and anti-biofilm activity even at low concentrations (0.15% to 0.4% HLE, v/v). Furthermore, the HLE disinfectant destabilized biofilm structures eradicated them due to the synergistic effect of EDTA and both antimicrobials (lactic acid and hydrogen peroxide), as revealed by confocal laser scanning microscopy. Additionally, sub-inhibitory concentrations of HLE disinfectant, with EDTA as an efflux pump inhibitor, inhibited the expression of multidrug EfrAB, NorE and MexCD efflux pumps in both planktonic and biofilm cultures. This could provide an alternative way to disinfect surfaces to avoid spreading multi-drug resistant strains in the food chain and the environment by decreasing efflux pump expression and consequently reducing the antibiotic selective pressure caused by systemic antibiotics and disinfectant use.

Proteomic analysis of Lactobacillus pentosus for the identification of potential markers involved in acid resistance and their influence on other probiotic features.

Acidity often prevents the undesirable microbial colonization both in fermented foods and under gastric conditions. Thus, the acid resistance of Lactobacillus pentosus strains used as starter cultures and/or probiotics requires further understanding. This was investigated by means of comparative proteomic approach using three strains representing the phenotypes: resistant (AP2-15), intermediate (AP2-18) and sensitive (LP-1) to acidic conditions. Proteomic analysis of constitutive phenotypes revealed that the intrinsic resistance of L. pentosus is associated with the over-production of three principal proteins: 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase 2 (PGAM-d), elongation factor G and 50S ribosomal protein L10, and additionally on ATP synthase subunit beta and chaperone protein DnaK; they are associated with metabolic pathways of proteins and carbohydrates, energy production and stress responses. Suggested protein biomarkers for acid resistance in L. pentosus include elongation factor G and PGAM-d, both being abundantly found in the constitutive proteome of the resistant phenotype under standard and acidic conditions. Furthermore, L. pentosus strains pre-exposed to acids displayed enhanced probiotic function such as auto-aggregation ability via surface proteins. We conclude that pre-exposure of probiotic L. pentosus strains to acid may strategically enhance their performance as starter cultures and probiotics.