Agar Spot Research Articles

In Vitro Inhibitory Effects and Co-Aggregation Activity of Lactobacilli on Candida albicans

Lactobacilli are considered important probiotics for the prevention of some infections. In this study, the antifungal effect of both cells and cell-free supernatants of twenty-three strains of lactobacilli were investigated against Candida albicans by co-aggregation, agar diffusion assay, agar spot assay and co-culture assay. In all cases, a fungistatic effect was recorded. In the agar diffusion assay and agar spot assay, an effect was established primarily for heterofermentative species via the production of lactic acid. The anti-Candida effect was higher with microbial suspension than with cultural supernatants in the co-culture assay. A strain-specific reduction in the yeast growth up to 28.9% in MRS broth and up to 17.1% in BHI broth was observed. Cells of Limosilactobacillus fermentum LLF-01 and Limosilactobacillus reuteri LLR-K67 showed the highest activity in both model systems. For all strains, a lower reduction up to 9.7% was recorded with cultural supernatants. L. fermentum LLF-01 showed the highest ability of co-aggregation (64.8%) with C. albicans, followed by Lactobacillus acidophilus LLA-01, Lactobacillus gasseri LLG-V74, Lactobacillus delbrueckii subsp. bulgaricus LLB-02 and two strains of Lactobacillus delbrueckii subsp. lactis LLL-14 and LLL-F18. The present study showed the potential of several strains of lactobacilli to affect the population of C. albicans in vitro. The combination of cultures with proved anti-Candida and co-aggregation activity in a probiotic formula may have a positive effect for the prevention of yeast overgrowth in the gut and hence for the suppression of candidiasis.

Sol-Gel Synthesis of Silica-Poly (Vinylpyrrolidone) Hybrids with Prooxidant Activity and Antibacterial Properties.

The present work deals with the sol-gel synthesis of silica-poly (vinylpyrrolidone) hybrid materials. The nanohybrids (Si-PVP) have been prepared using an acidic catalyst at ambient temperature. Tetramethyl ortosilane (TMOS) was used as a silica precursor. Poly (vinylpyrrolidone) (PVP) was introduced into the reaction mixture as a solution in ethanol with a concentration of 20%. The XRD established that the as-prepared material is amorphous. The IR and 29Si MAS NMR spectra proved the formation of a polymerized silica network as well as the hydrogen bonding interactions between the silica matrix and OH hydrogens of the silanol groups. The TEM showed spherical particle formation along with increased agglomeration tendency. The efficacy of SiO2/PVP nanoparticles as a potential antimicrobial agent against a wide range of bacteria was evaluated as bacteriostatic, using agar diffusion and spot tests. Combined effects of hybrid nanomaterial and antibiotics could significantly reduce the bactericidal concentrations of both the antibiotic and the particles, and they could also eliminate the antibiotic resistance of the pathogen. The registered prooxidant activity of the newly synthesized material was confirmative and explicatory for the antibacterial properties of the tested substance and its synergetic combination with antibiotics. The effect of new hybrid material on Crustacea Daphnia magna was also estimated as harmless under concentration of 0.1 mg/mL.

Comparative analysis of three methods for screening for colistin resistant <i>Escherichia coli</i>

The usage of colistin regarded as a drug of last resort has increased tremendously in recent years. This increase has been followed by an increase in the development of colistin resistant bacteria. Due the large size of colistin and its ability to adhere to plastic, the broth microdilution method using a special medium is the recommended testing method. Resource limited settings struggle with this method and employ alternate methods. This study therefore set out to determine colistin resistance in a group of Escherichia coli using three different methods comprised of colistin agar spot test (COL-AS), colistin drop test (COL-DT) and a disc diffusion method. A total of 51 Escherichia coli isolated from wound samples were screened for colistin resistance using the COLAS, COL-DT and colistin disc diffusion methods. Results showed a combined resistance rate of 96.1% among test isolates. Actual resistance rates varied between testing methods giving values of ranging from 37.3%, 66.0% and 88.2% for COL- DT, colistin disc diffusion methods and COL-A respectively. An assessment of test performance showed categorical agreement values and very major error values of 57.1%/36.7% for COL-DT and 63.3%/8.2% for COL-A. Results of this study show a high-level occurrence of colistin resistance among clinical Escherichia coli isolates. Furthermore, it demonstrates the superiority of the colistin agar test to the colistin drop test. It also points at a need to use higher concentrations of colistin in the screening tests.

Probiotic potential of Enterococcus strains with multiple enterocin-encoding genes

In this work, we sought to identify and characterize three strains of Enterococcus isolated from camel milk and fermented wheat, based on the capacity to produce bacteriocins and the probiotic potential. Polymerase chain reaction analyses were used to identify bacterial isolates and structural genes of bacteriocins, and also to detect potential enterococcal virulence genes (cylA, esp, gelE, efaAfs, hyl, ace, asa1, vanA, and vanB). The antimicrobial activity of the strains was investigated in solid media by the agar spot method against several pathogenic bacteria. The probiotic potential of the strains was also analysed using low pH (pH 3.0), bile salt resistance, DNAase, and antibiotic susceptibility assays. The strains were identified using rRNA16S sequencing gene, showing their belonging to E. faecium species. Based on PCR results, E. faecium CM9 and CM18 strains included in the genome the structural gene of enterocin A, enterocin B, and enterocin P, while E. faecium H3 possessed enterocin MR10A/B structural gene. The sequence analysis revealed that the H3 strain included in the genome the structural gene of enterocin L50A/B. All the pathogenic bacteria (Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes, E. coli, and Pseudomonas aeruginosa) were inhibited by the three strains E. faecium CM9, CM18, and H3. In the well diffusion test, the supernatants of the three strains exhibited inhibitory activity against Listeria monocytogenes CECT 4032. The strains showed high tolerance to low pH and bile salts, did not possess DNase, were susceptible to the majority of antibiotics assayed, and did not possess any of the virulence genes analyzed in this study. A promising candidate strains were identified as potential probiotics with anti-bacterial action against pathogenic bacteria.

Biocontrol of Escherichia coli O157:H7 by Enterobacter asburiae AEB30 on intact cantaloupe melons.

Escherichia coli O157:H7 causes >73,000 foodborne illnesses in the United States annually, many of which have been associated with fresh ready-to-eat produce including cantaloupe melons. In this study, we created a produce-associated bacterial (PAB) library containing >7500 isolates and screened them for the ability to inhibit the growth of E. coli O157:H7 using an invitro fluorescence-based growth assay. One isolate, identified by 16S and whole-genome sequence analysis as Enterobacter asburiae, was able to inhibit the growth of E. coli by ~30-fold invitro and produced zones of inhibition between 13 and 21 mm against 12 E. coli outbreak strains in an agar spot assay. We demonstrated that E. asburiae AEB30 was able to grow, persist and inhibit the growth of E. coli on cantaloupe melons under simulated pre- and post-harvest conditions. Analysis of the E. asburiae AEB30 genome revealed an operon encoding a contact-dependent growth inhibition (CDI) system that when mutated resulted in the loss of E. coli growth inhibition. These data suggest that E. asburiae AEB30 is a potential biocontrol agent to prevent E. coli contamination of cantaloupe melons in both pre- and post-harvest environments and that its mode of action is via a CDI system.

Exploring the Antifungal Activity of Various Natural Extracts in a Sustainable Saccharomyces cerevisiae Model Using Cell Viability, Spot Assay, and Turbidometric Microbial Assays

Saccharomyces cerevisiae is a sustainable yeast with many applications in the food industry. Here, we study the use of a Saccharomyces cerevisiae model composed of three different industrial strains (a wine, a beer and a baker’s strain) to assess the antifungal activity of three organic plant-based extracts (Hypericum perforatum 10% w/w, Pistacia lentiscus var. Chia 20% w/w and Rosmarinus officinalis 6% w/w). Three different methods were employed (agar disc diffusion, spot assay, and growth curve analysis). Only the Rosmarinus officinalis extract (6% w/w) exhibited inhibitory activity against all the tested yeast strains in the agar disc diffusion method. In the spot assay, all extracts and their carrier oils (sunflower oil and caprylic triglyceride) exerted similar mild antifungal activity. In the growth curve analysis, all extracts significantly lowered the growth rate of the yeasts, but this was not observed for the carrier oils. The results highlighted that it is important to consider more than one method for testing the antimicrobial activity of different compounds. The three yeast strains exhibited differences in their susceptibility to pharmaceutical antifungals, and the beer and baker’s yeasts were resistant to itraconazole. Moreover, polyphenols were detected in all natural extracts which may be linked to their antifungal activity. Our results suggest that we might consider multiple use of these natural extracts in the food industry as food additives or even preservatives to delay food spoilage.

Susceptibility to postbiotics - enterocins of methicillin-resistant Staphylococcus aureus strains isolated from rabbits

There is a major problem with the rising occurrence of highly virulent and multiply-resistant strains, including methicillin-resistant Staphylococcus aureus (MRSA), because of their difficult treatment. This study aimed to evaluate the antibacterial and antibiofilm effect of new enterocins (Ent) against potential pathogenic MRSA strains isolated from rabbits. Staphylococci were identified with PCR and screened for methicillin/oxacillin/cefoxitin resistance (MR) using the disk diffusion method and the PBP2’ Latex Agglutination Test Kit. Enzyme production, hemolysis, DNase activity, slime production, and biofilm formation were tested in MRSA strains. The susceptibility of MRSA to eight partially-purified enterocins (Ent) produced by E. faecium and E. durans strains was checked using agar spot tests. The antibiofilm activity of Ents was tested using a quantitative plate assay. Out of 14 MRSA, PBP testing confirmed MR in 8 strains. The majority of MRSA showed DNase activity and β-hemolysis. Slime production and moderate biofilm formation were observed in all strains. MRSA were susceptible to tested Ents (100–12,800 AU/mL; except Ent4231). The antibiofilm effect of Ents (except Ent4231) was noted in the high range (64.9–97.0%). These results indicate that enterocins offer a promising option for the prevention and treatment of bacterial infections caused by biofilm-forming MRSA.

Limonene synergistically augments fluconazole susceptibility in clinical Candida isolates from cleft lip and palate patients.

Cleft lip and palate (CLP) patients are prone to Candida infections (oral thrush) mainly due to poor oral hygiene, repetitive surgeries, and orthodontic procedures. This study was undertaken to evaluate the antifungal efficacy of limonene against clinical Candida isolates from CLP patients. The antifungal efficacy of limonene was studied alone and in combination with fluconazole (FLC) against six standards, twenty nine FLC sensitive, and three FLC resistant clinical strains using broth dilution, checkerboard microdilution, agar disk diffusion, growth curves, and spot assays. This nontoxic monoterpene gave low minimum inhibitory concentration (MIC) values of 300-375 µg/mL and 500-520 µg/mL for FLC susceptible and FLC resistant strains, respectively. It showed synergistic interaction with FLC in all clinical and standard Candida strains (fractional inhibitory concentration (FIC) index ≤0.5). Significant chemosensitization of FLC was observed even against resistant clinical isolates. Complete suppression of fungal growth was observed when using combinations. Negligible toxicity, easy availability, and potent antifungal properties suggest that limonene and FLC combinations in appropriate doses can make excellent antifungal mouthwashes during CLP treatment pre and post surgery. Impending in vivo studies are needed to validate the present data.

A Simple In Vitro Test to Select Stools for Fecal Microbiota Transplantation to Limit Intestinal Carriage of Extensively Drug-Resistant Bacteria.

Treatment options for multidrug-resistant bacterial infections are limited and often ineffective. Fecal microbiota transplantation (FMT) has emerged as a promising therapy for intestinal multidrug-resistant bacterial decolonization. However, clinical results are discrepant. The aim of our pilot study was to evaluate the screening performance of a simple diagnostic tool to select fecal samples that will be effective in decolonizing the intestine. Fecal samples from 10 healthy subjects were selected. We developed an agar spot test to evaluate their antagonistic activity toward the growth of VanA Enterococcus faecium and OXA-48-producing Klebsiella pneumoniae, two of the most serious and urgent threats of antibiotic resistance. Most fecal samples were able to limit the growth of both bacteria in vitro but with large inter-individual variation. The samples with the highest and lowest antagonistic activity were used for FMT in a mouse model of intestinal colonization. FMT was not successful in reducing intestinal colonization with VanA Enterococcus faecium, whereas FMT performed with the fecal sample showing the highest activity on the agar spot test was able to significantly reduce the intestinal colonization of mice with Klebsiella pneumoniae OXA-48. The agar spot test could thus serve as a reliable screening tool to select stool samples with the best potential to eradicate/reduce multidrug-resistant bacteria carriage after FMT.

Evaluation of anti-yeast metabolites produced by Lactobacillus strains and their potential application as bio-preservatives in traditional yogurt drink

Consumer demand for minimal food processing and reduced use of chemical preservatives has sparked interest in the use of biological preservatives. This study aimed to assess the anti-yeast effects of Lactobacillus delbrueckii, Limosilactobacillus fermentum, and Lactiplantibacillus plantarum strains on Saccharomyces cerevisiae, Candida albicans, and Geotrichum candidum. The anti-yeast activity of lactic acid bacteria (LAB) cell-free supernatant (CFS) was evaluated using well diffusion agar, agar spot assay, and scanning electron microscopy (SEM). Heat treatment's impact on the inhibitory capacity of CFS was also examined. Ion chromatography measured the production of propionic acid, lactic acid, and acetic acid in the supernatant. Furthermore, a yogurt drink containing the L. fermentum strain was produced. L. fermentum exhibited the highest anti-yeast activity against G. candidum, with an inhibition halo diameter of 16.60 mm. SEM revealed morphological changes in S. cerevisiae, C. albicans, and G. candidum following treatment with L. fermentum CFS. The production rates of lactic, propionic, and acetic acids in the L. fermentum supernatant without heat treatment were 94885.78, 115233, and 5936.02 ppm, respectively. Examination of mold, yeast, and coliform counts in samples inoculated with L. fermentum during 14 days of storage indicated that the microbial population remained within acceptable limits compared to control.

Lugdunin production and activity in Staphylococcus lugdunensis isolates are associated with its genotypes.

Lugdunin produced by Staphylococcus lugdunensis has been shown to have broad inhibitory activity against Gram-positive bacteria; however, lugdunin activity among S. lugdunensis isolates and its association with different agr, SCCmec, and sequence types remain unclear. We used matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to identify S. lugdunensis and collected 202 S. lugdunensis samples for further assays. Agar spot tests were performed to characterize S. lugdunensis lugdunin production and activity. Multilocus sequence typing, SCCmec, and agr genotyping were performed on S. lugdunensis. In all, 91 Staphylococcus aureus strains with varying vancomycin susceptibilities were used to examine lugdunin activity in S. lugdunensis. In total, 48 S. lugdunensis strains (23.8%) were found to be oxacillin-resistant S. lugdunensis (ORSL), whereas 154 (76.2%) were classified as oxacillin-sensitive S. lugdunensis (OSSL). Moreover, 16 (33.3%) ORSL and 35 (22.7%) OSSL strains showed antibacterial activity against S. aureus. Our data showed that most lugdunin-producing ORSL strains (14/48, 29.2%) were of ST3-SCCmec V-agr II genotypes, whereas most lugdunin-producing OSSL strains (15/154, 9.7%) were of ST3-agr II, followed by ST1-agr I (10/154, 6.5%). Our data also revealed that lugdunin exhibited weak inhibitory activity against the VISA ST239 isolate. In addition, we observed that ST239 VSSA was more resistant to lugdunin than ST5, ST59, and ST45 VSSA. Taken together, our data pioneered the epidemiology of lugdunin production in S. lugdunensis isolates and revealed its association with genotypes. However, further molecular and bioinformatics investigations are needed to elucidate the regulatory mechanisms of lugdunin production and activity. IMPORTANCE Lugdunin is active against both methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci by dissipating their membrane potential. However, the association of lugdunin activity with the genotypes of Staphylococcus lugdunensis has not been addressed. Here, we show the high prevalence of lugdunin-producing strains among ST1 (83.3%), ST2 (66.7%), and ST3 (53.3%) S. lugdunensis. Moreover, we identified the antibacterial activity of lugdunin-producing strains against VISA and hVISA. These results shed light on the potential application of lugdunin for the treatment of drug-resistant pathogens.

Predominance Biofilm Proteus mirabilis Isolates in the Skin Wound Infections, and Assessment Action of Antimicrobial Agents

Proteus mirabilis (P mirabilis) is the major pathogenic bacteria, an etiological agent of wound infections, particularly skin wound infections. Infection of wounds is the cause of morbidity and mortality on a universal scale. Different pathogens infecting wounds can colonize and multiply in the wounds, damaging the host tissue. This work aimed to determine biofilm formation, detect some virulence factors, and estimate the action of different antimicrobial agents i.e., antibiotics and probiotics Lactobacillus spp. P mirabilis was collected from different specimens of humans. Virulence factors of P mirabilis, especially atfA,rsbA, and mrpA, were observed in the isolates of P mirabilis through PCR technique. Isolates P mirabilis virulence factors were detected in more than 90%. The rate of susceptibility was 35 (94.6%) for cefoxitin, 29 (78.4%) for tobramycin, 22 (59.5%) for imipenem, followed by amikacin [16 (43.2%)], cefotaxime [11(29.7%)], ciprofloxacin [9 (24.3%)], ampicillin [1 (2.7%)]. As well as, different virulence biofilm genes related to the susceptibility for different antimicrobial agents, whether antibiotics or probiotics, such as rsbA [21 (100%)], atfA [20 (95.2%)] and mprA [19 (90.5%)]. Furthermore, probiotic lactobacilli spp. Such as Lactobacillus acidophilus and Lactobacillus casei shirota demonstrated high sensitivity against pathogens compared with antibiotics in different methods, such as agar spot (22-26 mm) and well diffusion (8-13 mm). In conclusion, P mirabilis demonstrated a high level of susceptibility to antibiotics against cefoxitin, tobramycin and imipenem and high resistance to ampicillin and others. Also, Lactobacillus spp. Demonstrated high sensitivity against pathogens compared with antibiotics. Keywords: Proteus mirabilis, biofilm, virulence factor genes, antibiotics, and probiotics

Biological Characterization and Metabolic Variations among Cell-Free Supernatants Produced by Selected Plant-Based Lactic Acid Bacteria.

The aim of this research was to assess the antibacterial and antioxidant properties as well as the variation in metabolites of the cell-free supernatant (CFS) produced by lactic acid bacteria (LAB) from local plants: Lactiplantibacillus plantarum ngue16, L. plantarum ng10, Enterococcus durans w3, and Levilactobacillus brevis w6. The tested strains exhibited inhibitory effects against pathogens, including Bacillus cereus, B. subtilis, Cronobacter sakazakii, Escherichia coli, Salmonella Typhimurium, and Staphylococcus aureus using the agar spot assay and well diffusion method. The CFS from all four strains displayed antibacterial activity against these pathogens with minimum inhibitory concentration (MIC) values ranging from 3.12 to 12.5 mg/mL and minimal bactericidal concentration (MBC) values ranging from 6.25 to 25.0 mg/mL. Moreover, the CFS demonstrated resilience within specific pH (3-8) and temperature (60-100 °C) ranges and lost its activity when treated with enzymes, such as Proteinase K and pepsin. Furthermore, the CFS exhibited antioxidant properties as evidenced by their ability to inhibit the formation of two radicals (1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) compared to the negative control, De Man, Rogosa, and Sharpe (MRS) broth. The use of proton-based nuclear magnetic resonance (1H-NMR) spectroscopy revealed the presence and quantification of 48 metabolites in both the CFS and MRS broths. Principal Component Analysis (PCA) effectively differentiated between CFS and MRS broth by identifying the specific metabolites responsible for the observed differences. The partial least squares (PLS) model demonstrated a significant correlation between the metabolites in the LAB supernatant and the tested antibacterial and antioxidant activities. Notably, anserine, GABA, acetic acid, lactic acid, uracil, uridine, propylene glycol, isopropanol, serine, histidine, and indol-3-lactate were identified as the compounds contributing the most to the highest antibacterial and antioxidant activities in the supernatant. These findings suggest that the LAB strains investigated have the potential to be utilized in the production of functional foods and the development of pharmaceutical products.

Isolation and Characterization of Pseudomonas aeruginosa Phages with a Broad Host Spectrum from Hospital Sewage Systems and Their Therapeutic Effect in a Mouse Model.

This study aimed to isolate and characterize phages as an alternative treatment of multidrug- or pan-drug-resistant Pseudomonas aeruginosa. Phage titers and bacterial densities correlated, with the phages disappearing after bacteria were eliminated. We isolated phages in filtered sewage water by a double-layered agar spot test. Fifty-eight P. aeruginosa strains were used to screen the host spectrum of the 14 phages isolated. Random amplification of polymorphic DNA-typing polymerase chain reaction was used to analyze the genomic homologies of the 58 host bacteria strains and four phages with a broad host spectrum. Transmission electron microscopy was used to observe the morphology of the four phages with a broad host spectrum. Mice with intraabdominal P. aeruginosa infection were used as an invivo animal model to investigate the therapeutic effect of the selected phage. Four virulent phages with a broad host spectrum specific to P. aeruginosa strains were isolated. They were all double-stranded DNA viruses and belonged to four different genotypes. The test curve showed that phage I had the highest adsorption rate, the shortest latent period, and the largest burst size. The infected mouse model indicated that small doses of phage I could prevent the death of infected mice. Phage titers and bacterial densities correlated, with phages disappearing after bacteria were eliminated. Phage I was the most effective and promising treatment of drug-resistant P. aeruginosa.

Peptide extract from spent yeast improves resistance of Saccharomyces cerevisiae to oxidative stress

Yeast cells face various stress factors during industrial fermentations, since they are exposed to harsh environmental conditions, which may impair biomolecules productivity and yield. In this work, the use of an antioxidant peptide extract obtained from industrial spent yeast was explored as supplement for Saccharomyces cerevisiae fermentation to prevent a common bottleneck: oxidative stress. For that, a recombinant yeast strain, producer of β-farnesene, was firstly incubated with 0.5 and 0.7 g/L peptide extract, in the presence and absence of hydrogen peroxide (an oxidative stress inducer), for 1–5 h, and then assayed for intracellular reactive oxygen species, and growth ability in agar spot assays. Results showed that under 2 mM H2O2, the peptide extract could improve cells growth and reduce reactive oxygen species production. Therefore, this antioxidant effect was further evaluated in shake-flasks and 2-L bioreactor batch fermentations. Peptide extract (0.7 g/L) was able to increase yeast resistance to the oxidative stress promoted by 2 mM H2O2, by reducing reactive oxygen species levels between 1.2- and 1.7-fold in bioreactor and between 1.2- and 3-fold in shake-flask fermentations. Moreover, improvements on yeast cell density of up to 1.5-fold and 2-fold, and on biomolecule concentration of up to 1.6-fold and 2.8-fold, in bioreactor and shake-flasks, respectively, were obtained. Thus, culture medium supplementation with antioxidant peptide extracted from industrial spent yeast is a promising strategy to improve fermentation performance while valuing biomass waste. This valorization can promote a sustainable and eco-friendly solution for the biotechnology industry by the implementation of a circular economy model.Key points• Peptide extract from spent yeast applied for the first time on yeast fermentation.• Antioxidant peptide extract enhanced S. cerevisiae oxidative stress resistance.• Fermentation performance under stress improved by peptide extract supplementation.

In Vitro Evaluation of Commercial Probiotic Products Containing Streptococcus salivarius K12 by Assessment of Probiotic Viability and Inhibitory Potency against Respiratory Pathogens

Upper respiratory infections (URI) are the most frequent illnesses, especially in children. The majority of those infections are prescribed broad-spectrum antibiotics, which are associated with various side effects and with the increase in multi-drug-resistant strains. A promising alternative approach is the administration of the probiotic strain Streptococcus salivarius K12 (SSK12) that colonizes the upper respiratory tract (URT) and produces the salivaricins A2 and B, which strongly antagonize the growth of key respiratory pathogens. However, since for food supplements no quality controls of the active probiotic ingredient are mandatory, the efficacy of commercial products containing SSK12 may vary. This study aimed to investigate the in vitro efficacy of several commercial SSK12-containing probiotics, positioned for the prevention of respiratory infections. The parameters evaluated to determine the in vitro efficacy included the viability of the probiotic bacterial strain and the minimum inhibitory dilution (MID) of the probiotic, determined by the agar spot method, against the pathogenic/potential pathogenic bacterial strains Streptococcus pyogenes FF22 and Micrococcus luteus T18. All tests were carried out both 12 and 24 months after manufacturing (AM) for each commercial product. The viability ranged from 9 × 108 to 4.4 × 109 CFU/serving at 12 months AM and from 8.5 × 107 to 2.8 × 109 CFU/serving at 24 months AM. The MID was, in general, positively correlated with the probiotic bacterium viability and varied between the commercial products, ranging from 10−5 to 10−7 at 12 months AM and from 10−4 to 10−7 at 24 months AM. Moreover, the inhibition zones related to the two indicator strains were variable in diameter for different products. The high variation of the in vitro efficacy of commercial products containing SSK12 may explain the different results reported in the literature regarding the clinical benefits of these preparations, and the determination of this parameter may be useful to evaluate the quality of probiotic products containing this bacterial strain.

Antimicrobial Activities and Biopreservation Potential of Lactic Acid Bacteria (LAB) from Raw Buffalo (Bubalus bubalis) Milk.

The aim of this study was to investigate the antimicrobial and biopreservation potential of lactic acid bacteria. The potential probiotic culture inhibited the growth of gram-positive and gram-negative foodborne pathogens in agar spot assay with inhibition zones ranging from 10 to 21 mm in diameter. The strains showed coaggregation capabilities ranging from 7 to 71% with tested food pathogens including Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica subsp. enterica serovar Typhimurium. The effect of cell-free supernatants on the release of 260 nm absorbing material, especially nucleic acids, was evaluated and indicated the antagonistic activity on foodborne pathogens, the highest being Lactobacillus paraplantarum against E. coli (3.77) and S. aureus (3.86) after 60 min. The effect of cell-free supernatant (CFS) on the growth of pathogens showed that Lactobacillus paraplantarum 11 and L. pentosus 93 had the highest inhibitory activity against tested strains. The biopreservation assay indicated that the potential probiotic strains Lactobacillus paraplantarum 11 (BT), Lactiplantibacillus plantarum 19, Lactobacillus pentosus 42, Limosilactobacillus fermentum 60, Lactobacillus pentosus 93, and Limosilactobacillus reuteri 112 were effective in reducing the Listeria monocytogenes population in raw buffalo milk. Complete Listeria monocytogenes inhibition was observed after 6-8 days. This study showed that probiotic LAB from buffalo milk have antimicrobial and biopreservation potential; these strains have the potential to be utilized as biopreservative agents in food products.

Antimicrobial Activity of Ligilactobacillus animalis SWLA-1 and Its Cell-Free Supernatant against Multidrug-Resistant Bacteria and Its Potential Use as an Alternative to Antimicrobial Agents.

The emergence of multidrug-resistant (MDR) bacteria and the spread of antimicrobial resistance among various bacteria are major threats to the global community. Due to the increased failure of classical antibiotic treatments against MDR bacterial infections, probiotics and their antimicrobial compounds have been suggested as promising alternatives to deal with MDR bacteria. Various strains of lactic acid bacteria have been reported to produce antagonistic molecules against pathogens. A new strain of Ligilactobacillus animalis, L. animalis SWLA-1, isolated from the feces of healthy dogs, shows strong antimicrobial activity against not only common pathogens but also MDR bacteria. In this study, we compared the antimicrobial activity of L. animalis SWLA-1 with that of other lactobacilli and antibiotics using an agar spot assay. Additionally, a novel spot inhibition index was developed and validated to quantitively evaluate the inhibitory activities of lactobacilli and antibiotics. A competitive coculture assay of L. animalis SWLA-1 with MDR bacteria further demonstrated its antibacterial activity. Furthermore, we evaluated the antimicrobial activity of the cell-free supernatant (CFS) of L. animalis SWLA-1 and its stability under various conditions in vitro. We found that L. animalis SWLA-1 and its CFS are potential alternatives to classic antimicrobial agents.

The Antimicrobial Effect of Various Single-Strain and Multi-Strain Probiotics, Dietary Supplements or Other Beneficial Microbes against Common Clinical Wound Pathogens.

The skin is the largest organ in the human body and is colonized by a diverse microbiota that works in harmony to protect the skin. However, when skin damage occurs, the skin microbiota is also disrupted, and pathogens can invade the wound and cause infection. Probiotics or other beneficial microbes and their metabolites are one possible alternative treatment for combating skin pathogens via their antimicrobial effectiveness. The objective of our study was to evaluate the antimicrobial effect of seven multi-strain dietary supplements and eleven single-strain microbes that contain probiotics against 15 clinical wound pathogens using the agar spot assay, co-culturing assay, and agar well diffusion assay. We also conducted genera-specific and species-specific molecular methods to detect the DNA in the dietary supplements and single-strain beneficial microbes. We found that the multi-strain dietary supplements exhibited a statistically significant higher antagonistic effect against the challenge wound pathogens than the single-strain microbes and that lactobacilli-containing dietary supplements and single-strain microbes were significantly more efficient than the selected propionibacteria and bacilli. Differences in results between methods were also observed, possibly due to different mechanisms of action. Individual pathogens were susceptible to different dietary supplements or single-strain microbes. Perhaps an individual approach such as a 'probiogram' could be a possibility in the future as a method to find the most efficient targeted probiotic strains, cell-free supernatants, or neutralized cell-free supernatants that have the highest antagonistic effect against individual clinical wound pathogens.

Exploring the role of antimicrobials in the selective growth of purple phototrophic bacteria through genome mining and agar spot assays.

Purple non-sulphur bacteria (PNSB) are an emerging group of microbes attractive for applied microbiology applications such as wastewater treatment, plant biostimulants, microbial protein, polyhydroxyalkanoates and H2 production. These photoorganoheterotrophic microbes have the unique ability to grow selectively on organic carbon in anaerobic photobioreactors. This so-called selectivity implies that the microbial community will have a low diversity and a high abundance of a particular PNSB species. Recently, it has been shown that certain PNSB strains can produce antimicrobials, yet it remains unclear whether these contribute to competitive inhibition. This research aimed to understand which type of antimicrobial PNSB produce and identify whether these compounds contribute to their selective growth. Mining 166 publicly-available PNSB genomes using the computational tool BAGEL showed that 59% contained antimicrobial encoding regions, more specifically biosynthetic clusters of bacteriocins and non-ribosomal peptide synthetases. Inter- and intra-species inhibition was observed in agar spot assays for Rhodobacter blasticus EBR2 and Rhodopseudomonas palustris EBE1 with inhibition zones of, respectively, 5.1 and 1.5-5.7 mm. Peptidomic analysis detected a peptide fragment in the supernatant (SVLQLLR) that had a 100% percentage identity match with a known non-ribosomal peptide synthetase with antimicrobial activity.