Kinetic Analysis of Bifidobacterial Metabolism Reveals a Minor Role for Succinic Acid in the Regeneration of NAD+ through Its Growth-Associated Production

Several strains belonging to the genus Bifidobacterium were tested to determine their abilities to produce succinic acid. Bifidobacterium longum strain BB536 and Bifidobacterium animalis subsp. lactis strain Bb 12 were kinetically analyzed in detail using in vitro fermentations to obtain more insight into the metabolism and production of succinic acid by bifidobacteria. Changes in end product formation in strains of Bifidobacterium could be related to the specific rate of sugar consumption. When the specific sugar consumption rate increased, relatively more lactic acid and less acetic acid, formic acid, and ethanol were produced, and vice versa. All Bifidobacterium strains tested produced small amounts of succinic acid; the concentrations were not more than a few millimolar. Succinic acid production was found to be associated with growth and stopped when the energy source was depleted. The production of succinic acid contributed to regeneration of a small part of the NAD⁺, in addition to the regeneration through the production of lactic acid and ethanol.     

In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids

The aim of this study was to investigate the antimicrobial properties of fifteen selected strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera against Gram-positive and Gram-negative pathogenic bacteria.In vitro antibacterial activity was initially investigated by an agar spot method. Results from the agar spot test showed that most of the selected strains were able to produce active compounds on solid media with antagonistic properties against Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Clostridium difficile. These results were also confirmed when cell-free culture supernatants (CFCS) from the putative probiotics were used in an agar well diffusion assay. Neutralization of the culture supernatants with alkali reduced the antagonistic effects. These experiments are able to confirm the capacity of potential probiotics to inhibit selected pathogens. One of the main inhibitory mechanisms may result from the production of organic acids from glucose fermentation and consequent lowering of culture pH. This observation was confirmed when the profile of organic acids was analysed demonstrating that lactic and acetic acid were the principal end products of probiotic metabolism.Furthermore, the assessment of the haemolytic activity and the susceptibility of the strains to the most commonly used antimicrobials, considered as basic safety aspects, were also studied.The observed antimicrobial activity was mainly genus-specific, additionally significant differences could be observed among species.     

Antimicrobial activity of chemically and biologically synthesized silver nanoparticles against some fish pathogens

Pathogens isolated from fish appear to possess considerable antimicrobial resistance and represent a problem for the economy and public health. Natural antimicrobial substitutes to traditional antibiotics represent an essential tool in the fight against antibiotic resistance. Nanotechnology has shown considerable potential in different research fields, and the antimicrobial properties of silver nanoparticles are known. Silver has been used for medical purposes since ancient times because of its bactericidal properties, and the highly reactive surfaces of silver nanoparticles (AgNPs) indicate that they might have a function in antimicrobial applications. This work aimed to study the antimicrobial properties of biologically produced AgNPs from Origanum vulgare leaves compared to chemically produced AgNPs. Both types were characterized by UV–vis spectrophotometry, TEM, and dynamic light scattering and tested against three bacterial strains (Streptococcus agalactiae, and Aeromonas hydrophila, both isolated from Nile tilapia and Vibrio alginolyticus, isolated from sea bass) and three fungal strains (Aspergillus flavus, Fusarium moniliforme, and Candida albicans, all isolated from Nile tilapia). Disk diffusion test and evaluation of ultrastructure changes of tested microorganisms treated with AgNPs by transmission electron microscopy were performed. Moreover, the hemolytic properties of AgNPs were studied on chicken and goat red blood cells. The results obtained declare that the green biological production of silver nanoparticles is safer and more effective than the chemical one; moreover, AgNPs have interesting dose-dependent antimicrobial properties, with better results for biologically produced ones; their effectiveness against tested bacterial and fungal strains opens the way to their use to limit fish diseases, increase economy and improve human health.     

The Effect of Various Probiotic Strains or Avilamycin Feed Additive on Immune Defense Markers and Acute-Phase Response to Salmonella Infection in Chickens

Probiotics are a nutritional tool for disease prevention. It has been proposed that stimulation of immune response could affect the growth-promoting properties of antimicrobial growth promoters as well as the control of foodborne pathogens. The current study compares immune response in the blood of 280 non-infected and Salmonella-infected chickens fed either with the growth promoter avilamycin or with one of five probiotic strains of Lactobacillus and Bifidobacterium, which also showed growth-promoting properties. All of the probiotic strains stimulated superoxide anion production and the proliferation of leukocytes, while raising lysozyme and γ-globulin levels (by up to 65%, p < 0.01), which are important factors in native and cell-mediated immune defense against pathogens. In contrast, among the two strains examined, specific Salmonella antibodies were induced only by L. salivarius, and not by B. animalis, as assessed by the ELISA method and confirmed by an agglutination reaction (p < 0.05). In the avilamycin-fed group, both non-infected and infected chickens showed decreased levels of these immune markers (by 30%) and increased levels of ceruloplasmin by up to 35%. In contrast, the probiotics suppressed acute-phase response assessed by ceruloplasmin by up to 32%. This correlation implies that various antimicrobial feed additives have a distinct effect on immunomodulation, which may affect different mechanisms in the nutrition-related metabolism associated with the rate of weight gain in chickens. The data could contribute to the design of innovative antimicrobial feed additives in the food industry and consequently to well-being of humans.     

Gut bacterium of Dendrobaena veneta (Annelida: Oligochaeta) possesses antimycobacterial activity

The new bacterial strain with antimycobacterial activity has been isolated from the midgut of Dendrobaena veneta (Annelida). Biochemical and molecular characterization of isolates from 18 individuals identified all as Raoultella ornithinolytica genus with 99% similarity. The bacterium is a possible symbiont of the earthworm D. veneta. The isolated microorganism has shown the activity against four strains of fast-growing mycobacteria: Mycobacterium butiricum, Mycobacterium jucho, Mycobacterium smegmatis and Mycobacterium phlei. The multiplication of the gut bacterium on plates with Sauton medium containing mycobacteria has caused a lytic effect. After the incubation of the cell free extract prepared from the gut bacterium with four strains of mycobacteria in liquid Sauton medium, the cells of all tested strains were deformed and divided to small oval forms and sometimes created long filaments. The effect was observed by the use of light, transmission and scanning microscopy. Viability of all examined species of mycobacteria was significantly decreased. The antimycobacterial effect was probably the result of the antibiotic action produced by the gut bacterium of the earthworm. The application of ultrafiltration procedure allowed to demonstrate that antimicrobial substance with strong antimycobacterial activity from bacterial culture supernatant, is a protein with the molecular mass above 100 kDa.     

Mining the Microbiota of the Neonatal Gastrointestinal Tract for Conjugated Linoleic Acid-Producing Bifidobacteria

This study was designed to isolate different strains of the genus Bifidobacterium from the fecal material of neonates and to assess their ability to produce the cis-9, trans-11 conjugated linoleic acid (CLA) isomer from free linoleic acid. Fecal material was collected from 24 neonates aged between 3 days and 2 months in a neonatal unit (Erinville Hospital, Cork, Ireland). A total of 46 isolates from six neonates were confirmed to be Bifidobacterium species based on a combination of the fructose-6-phosphate phosphoketolase assay, RAPD [random(ly) amplified polymorphic DNA] PCR, pulsed-field gel electrophoresis (PFGE), and partial 16S ribosomal DNA sequencing. Interestingly, only 1 of the 11 neonates that had received antibiotic treatment produced bifidobacteria. PFGE after genomic digestion with the restriction enzyme XbaI demonstrated that the bifidobacteria population displayed considerable genomic diversity among the neonates, with each containing between one and five dominant strains, whereas 11 different macro restriction patterns were obtained. In only one case did a single strain appear in two neonates. All genetically distinct strains were then screened for CLA production after 72 h of incubation with 0.5 mg of free linoleic acid ml⁻¹ by using gas-liquid chromatography. The most efficient producers belonged to the species Bifidobacterium breve, of which two different strains converted 29 and 27% of the free linoleic acid to the cis-9, trans-11 isomer per microgram of dry cells, respectively. In addition, a strain of Bifidobacterium bifidum showed a conversion rate of 18%/μg dry cells. The ability of some Bifidobacterium strains to produce CLA could be another human health-promoting property linked to members of the genus, given that this metabolite has demonstrated anticarcinogenic activity in vitro and in vivo.     

Phylogenetic Analysis of the Genus Bifidobacterium and Related Genera Based on 16S rDNA Sequences

The 16S rRNA gene sequences were determined for type strains of 21 Bifidobacterium species. A phylogenetic tree was constructed using the determined sequences and sequences from DNA databases, which contain the sequences of 11 type strains of Bifidobacterium species and 11 strains of related genera. All species of the genus Bifidobacterium and Gardnerella vaginalis ATCC 14018 belonged to a cluster phylogenetically distinct from the other genera. The cluster was divided into two subclusters: subcluster 1 composed of most species of Bifidobacterium and G. vaginalis, and subcluster 2 consisting of two species, B. denticolens and B. inopinatum; both of which were isolated from human dental caries. In the genus Bifidobacterium, four groups of species are known to be moderately to highly related by DNA-DNA hybridization. The four groups of species exhibited more than 99% similarity among their 16S rDNA sequences within each group. These results indicated that species with around 99% or more similarity in their 16S rDNA sequences should be confirmed for species identities.     

Bacteriocin production and gene sequencing analysis from vaginal Lactobacillus strains

The human vagina is a complex and dynamic ecosystem containing an abundance of microorganisms. In women of childbearing age, this system is dominated by Lactobacillus spp. In the present work, seventeen newly isolated vaginal strains were identified by 16S rDNA sequencing and were investigated for their antimicrobial properties. Twelve of the isolated Lactobacillus strains showed activity against one or more microorganisms. Six and five of them produced substances that inhibited the growth of two different Klebsiella strains and Staphylococcus aureus, respectively. Two lactobacilli strains were active against an Escherichia coli strain, one isolate was active against an Enterococus faecalis strain and another lactobacilli strain showed antimicrobial activity against a Candida parapsilosis strain. The nature of the active compounds was additionally studied, and the presence of bacteriocin-like substances was proved. The genes related to the bacteriocin production in three of the newly isolated strains were identified and sequenced. The presence of gassericin A operon in the genome of the species Lactobacillus crispatus was described for the first time. The presence of antimicrobial activity contributes to their possible use as potential probiotic strains after further research.     

Antimicrobial peptides produced by <Emphasis Type="Italic">Brevibacillus</Emphasis> spp.: structure,classification and bioactivity: a mini review

Species that are currently listed under the genus Brevibacillus (formerly, Bacillus brevis cluster) have been a rich source of antimicrobial peptides for many decades. The first known peptide antibiotic, gramicidin, is presumed to be produced by a Brevibacillus sp. Members of the genus are widely spread in nature. They can be found in a variety of environments including intestinal tracts of animals, seawater, and soil. Some Brevibacillus strains have been used commercially as probiotics. Bioactive peptides produced by Brevibacillus spp. include antibacterial, antifungal and anti-invertebrate agents. Brevibacillus antimicrobial peptides are synthesized through ribosomal or nonribosomal pathway; these two groups can be further categorized based on specific structural features such as cyclization and presence of lipid chain. Some of the antimicrobial compounds produced by this genus share structural similarities that were overlooked previously. For example, the structural similarity between BT peptide, brevibacillin, and bogorol was revealed only recently. Here we review and classify Brevibacillus antimicrobial peptides and summarize their bioactivities and potential applications.     

The role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines

Amphibian species have experienced population declines and extinctions worldwide that are unprecedented in recent history. Many of these recent declines have been linked to a pathogenic skin fungus, Batrachochytrium dendrobatidis, or to iridoviruses of the genus Ranavirus. One of the first lines of defense against pathogens that enter by way of the skin are antimicrobial peptides synthesized and stored in dermal granular glands and secreted into the mucus following alarm or injury. Here, I review what is known about the capacity of amphibian antimicrobial peptides from diverse amphibians to inhibit B. dendrobatidis or ranavirus infections. When multiple species were compared for the effectiveness of their in vitro antimicrobial peptides defenses against B. dendrobatidis, non-declining species of rainforest amphibians had more effective antimicrobial peptides than species in the same habitat that had recently experienced population declines. Further, there was a significant correlation between the effectiveness of the antimicrobial peptides and resistance of the species to experimental infection. These studies support the hypothesis that antimicrobial peptides are an important component of innate defenses against B. dendrobatidis. Some amphibian antimicrobial peptides inhibit ranavirus infections and infection of human T lymphocytes by the human immunodeficiency virus (HIV). An effective antimicrobial peptide defense against skin pathogens appears to depend on a diverse array of genes expressing antimicrobial peptides. The production of antimicrobial peptides may be regulated by signals from the pathogens. However, this defense must also accommodate potentially beneficial microbes on the skin that compete or inhibit growth of the pathogens. How this delicate balancing act is accomplished is an important area of future research.     

A basidiomycetous yeast, Pseudozyma crassa, produces novel diastereomers of conventional mannosylerythritol lipids as glycolipid biosurfactants

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These compounds show not only excellent surface-active properties, but also versatile biochemical actions. During a survey of new MEL producers, we found that a basidiomycetous yeast, Pseudozyma crassa, extracellularly produces three glycolipids. When glucose and oleic acid were used as the carbon source, the total amount of glycolipids reached approximately 4.6 g/L in the culture medium. The structures of these glycolipids were similar to those of well-known MEL-A, -B, and -C, respectively. Very interestingly, in all the present glycolipids, the configuration of the erythritol moiety was entirely opposite to that of conventional MELs. The present glycolipids were identified to have the carbohydrate structure of 4-O-β-d-mannopyranosyl-(2R,3S)-erythritol, stereochemically different from 4-O-β-d-mannopyranosyl-(2S,3R)-erythritol of conventional MELs. Furthermore, these new glycolipids possessed both short-chain acids (C₂ or C₄) and long-chain acids (C₁₄, C₁₆, or C₁₈) on the mannose moiety. The major component of the present glycolipids clearly showed different interfacial and biological properties, compared to conventional MELs comprising two medium-chain acids on the mannose moiety. Accordingly, the novel MEL diastereomers produced by P. crassa should provide us with different glycolipid functions, and facilitate a broad range of applications of MELs.     

Active metabolites produced by Penicillium chrysogenum IFL1 growing on agro-industrial residues

Microbial extracts continue to be a productive source of new molecules with biotechnological importance. Fungi of the genus Penicillium are known to produce biologically active secondary metabolites. The goal of this work is verify the production of antimicrobial metabolites by Penicillium chrysogenum IFL1 using agro-industrial residues. P. chrysogenum IFL1 produced active metabolites growing on the agro-industrial residues, grape waste and cheese whey. The 7-day cultures showed antimicrobial activities against bacteria, fungi and amoebae. The filtrate of the cheese whey culture inhibited the growth of the bacteria Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa, the fungus Fusarium oxysporum and the amoeba Acanthamoeba polyphaga. Due to the greater antimicrobial activity of the cheese whey culture, a footprinting profile was carried out using the ESI-MS and ESI-MS/MS techniques. The presence of penicillin G and other metabolites that have antimicrobial activity such as penicillin V and rugulosin can be suggested. P. chrysogenum IFL1 was able to produce a wide variety of antimicrobial compounds on agro-industrial residues, which makes the process ecologically friendly.     

Bioactive polar natural compounds from garlic and onions

Bioactive natural compounds from garlic and onions have been the focus of researches for decades, firstly due to their pharmacological effects, and secondly due to their defence properties against plant diseases. In fact, garlic and onion, belonging to Allium genus, are among the oldest food plants known since ancient times and used as ingredient of many recipes and for therapeutic properties. These plants are well known to produce bioactive apolar sulphur compounds but less is known about their polar natural compounds, such as phenols, sapogenins and saponins, that are more stable to cooking, So, we continued our work on the discovery of polar bioactive metabolites from Allium with the isolation of a number of sapogenins and saponins from the wild onion species Allium elburzense, Allium hirtifolium, Allium atroviolaceum, and Allium minutiflorum, and, more recently, from the cultivated white onion, Allium cepa, and garlic, Allium sativum. In particular, the sapogenins and saponins isolated from A. elburzense and A. hirtifolium, named elburzensosides and hirtifoliosides respectively, exhibited significant antispasmodic properties. In addition, the saponins named minutosides isolated from A. minutiflorum showed promising antimicrobial activity. More recently the phytochemical analysis of A. cepa and A. sativum has been undertaken and afforded the characterization of saponins, phenols and N-cynnamic amides which showed significant antifungal activity.     

Structure stability/activity relationships of sulfone stabilized N,N-dichloroamines

Structure stability/activity relationships (SXR) of a new class of N,N-dichloroamine compounds were explored to improve antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans while maintaining aqueous solution stability. This study identified a new class of solution-stable and topical antimicrobial agents. These agents are sulfone-stabilized and possess either a quaternary ammonium or sulfonate appendages as a water solubilizing group. Several unique challenges were confronted in the synthesis of these novel compounds which are highlighted in the discussion.     

Comparative Genomic Analysis of 45 Type Strains of the Genus Bifidobacterium: A Snapshot of Its Genetic Diversity and Evolution

Bifidobacteria are well known for their human health-promoting effects and are therefore widely applied in the food industry. Members of the Bifidobacterium genus were first identified from the human gastrointestinal tract and were then found to be widely distributed across various ecological niches. Although the genetic diversity of Bifidobacterium has been determined based on several marker genes or a few genomes, the global diversity and evolution scenario for the entire genus remain unresolved. The present study comparatively analyzed the genomes of 45 type strains. We built a robust genealogy for Bifidobacterium based on 402 core genes and defined its root according to the phylogeny of the tree of bacteria. Our results support that all human isolates are of younger lineages, and although species isolated from bees dominate the more ancient lineages, the bee was not necessarily the original host for bifidobacteria. Moreover, the species isolated from different hosts are enriched with specific gene sets, suggesting host-specific adaptation. Notably, bee-specific genes are strongly associated with respiratory metabolism and are potential in helping those bacteria adapt to the oxygen-rich gut environment in bees. This study provides a snapshot of the genetic diversity and evolution of Bifidobacterium, paving the way for future studies on the taxonomy and functional genomics of the genus.     

In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.     

Comparative genomics of Bifidobacterium species isolated from marmosets and humans

The genus Bifidobacterium is purported to have beneficial consequences for human health and is a major component of many gastrointestinal probiotics. Although species of Bifidobacterium are generally at low relative frequency in the adult human gastrointestinal tract, they can constitute high proportions of the gastrointestinal communities of adult marmosets. To identify genes that might be important for the maintenance of Bifidobacterium in adult marmosets, ten strains of Bifidobacterium were isolated from the feces of seven adult marmosets, and their genomes were sequenced. There were six B. reuteri strains, two B. callitrichos strains, one B. myosotis sp. nov. and one B. tissieri sp. nov. among our isolates. Phylogenetic analysis showed that three of the four species we isolated were most closely related to B. bifidum, B. breve and B. longum, which are species found in high abundance in human infants. There were 1357 genes that were shared by at least one strain of B. reuteri, B. callitrichos, B. breve, and B. longum, and 987 genes that were found in all strains of the four species. There were 106 genes found in B. reuteri and B. callitrichos but not in human bifidobacteria, and several of these genes were involved in nutrient uptake. These pathways for nutrient uptake appeared to be specific to Bifidobacterium from New World monkeys. Additionally, the distribution of Bifidobacterium in fecal samples from captive adult marmosets constituted as much as 80% of the gut microbiome, although this was variable between individuals and colonies. We suggest that nutrient transporters may be important for the maintenance of Bifidobacterium during adulthood in marmosets.     

Monascus secondary metabolites: production and biological activity

The genus Monascus, comprising nine species, can reproduce either vegetatively with filaments and conidia or sexually by the formation of ascospores. The most well-known species of genus Monascus, namely, M. purpureus, M. ruber and M. pilosus, are often used for rice fermentation to produce red yeast rice, a special product used either for food coloring or as a food supplement with positive effects on human health. The colored appearance (red, orange or yellow) of Monascus-fermented substrates is produced by a mixture of oligoketide pigments that are synthesized by a combination of polyketide and fatty acid synthases. The major pigments consist of pairs of yellow (ankaflavin and monascin), orange (rubropunctatin and monascorubrin) and red (rubropunctamine and monascorubramine) compounds; however, more than 20 other colored products have recently been isolated from fermented rice or culture media. In addition to pigments, a group of monacolin substances and the mycotoxin citrinin can be produced by Monascus. Various non-specific biological activities (antimicrobial, antitumor, immunomodulative and others) of these pigmented compounds are, at least partly, ascribed to their reaction with amino group-containing compounds, i.e. amino acids, proteins or nucleic acids. Monacolins, in the form of β-hydroxy acids, inhibit hydroxymethylglutaryl-coenzyme A reductase, a key enzyme in cholesterol biosynthesis in animals and humans.     

Solid-phase microextraction of volatile compounds from flowers of two Brunfelsia species

Two plant species belonging to the genus Brunfelsia, commonly known as “Yesterday-Today-and-Tomorrow”, are closely related, however, differ by their flower fragrance. Flowers of Brunfelsia australis present a pleasant fragrance, whereas flowers of Brunfelsia pauciflora are scentless. SPME/GC/MS analysis on flower samples of both species of Brunfelsia indicated that flowers of B. australis emitted a fresh flowery fragrance, essentially comprising two monoterpenic compounds (linalool and (E)-ocimene). B. pauciflora, on the other hand, produced only a few sesquiterpenoids. These results are considered in an ecological and evolutionary context.     

Phytochemicals,mineral contents,antioxidants, and antimicrobial activities of propolis produced by Brunei stingless bees Geniotrigona thoracica,Heterotrigona itama,and Tetrigona binghami

The present study focused on the evaluation of phytochemical properties, essential mineral elements, and heavy metals contained in raw propolis produced by stingless bees Geniotrigona thoracica, Heterotrigona itama, and Tetrigona binghami found in the same ecological conditions and environment in Brunei Darussalam. The results indicated that propolis of the three stingless bee species mainly consisted of lipids (45.60–47.86%) and very low carbohydrate (0.17–0.48%) and protein contents (0.18–1.18%). The propolis was rich in mineral elements, thus good sources of minerals, while they contained low concentrations of all heavy metals. Propolis of the different bee species could be distinguished based on their mineral compositions. The vibrational and absorption spectra suggested that propolis contains π-conjugated aliphatic and aromatic compounds as well as aromatic acids having amine, ester, carbonyl, alkyl, and hydroxyl functional groups which might be attributed to the presence of phenolic and flavonoid compounds. The antioxidant capacity of the propolis, based on radical scavenging activity of their ethanol extract, was in line with their total phenolic content. The ethanol extract of the propolis also showed antimicrobial activities against four bacterial strains (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa). The propolis showed slightly higher antibacterial activity against Gram-positive (B. subtilis and S. aureus) bacteria, indicating that the antimicrobial active compounds could be associated with flavonoids, which were quantified to be approximately comparable in all the propolis.