Bacteriostatic Activity Research Articles

Deciphering the Mechanism of Action of a Short, Synthetic Designer AMP Against Gram-Negative Bacteria.

Antimicrobial peptides (AMPs), produced in various organisms, including plants, as a first line of defense, are potent, functionally versatile, fast-acting small peptides with a net charge and diverse structures. Most AMPs demonstrate potent antibacterial activity, and AMPs with multimodal actions can potentially delay the development of antimicrobial resistance (AMR), one of the top 10 global public health challenges categorized by the WHO. Notably, the FDA has already approved several AMPs (Mol. Wt. ≤ 2 kDa) as antibiotics; however, there are not enough new-age antibiotics in the current pipeline to combat the looming problem of AMR in the clinic. Nevertheless, despite their potential, natural AMPs have their fair share of shortcomings for straightforward therapeutic applications. Therefore, extensive research on developing designer synthetic AMPs with broad-spectrum antimicrobial activity is currently being undertaken to mitigate the AMR challenge. In this context, we recently demonstrated a short synthetic designer AMP (SR17: ≤ 16 aa, mol. Wt. ≤ 2 kDa) that exhibits broad-spectrum bacteriostatic and bactericidal action against both gram-negative (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii) and gram-positive (Staphylococcus aureus) bacteria. Interestingly, in gram-negative bacteria, the outer membrane proteins (OMPs) play a key role in transporting nutrients like iron from their surroundings through siderophores, which play a crucial role in various biochemical processes essential for their survival and growth. In the current study, the ability of SR17 to target the iron-transporting OMPs acting as the siderophore uptake system is investigated through computational techniques. A series of docking and molecular dynamics (MD) simulation studies involving iron transporters of various gram-negative bacteria indicate that SR17 can occupy the binding pocket in the OMPs necessary for binding of the iron-chelated siderophores, which is likely to prevent the further uptake of siderophores, affecting the growth and survival of the bacteria. Additionally, SR17 may potentially reach the bacterial cytoplasm by utilizing the siderophore uptake system and disrupt essential cytoplasmic processes, leading to the death of the bacteria, as observed in experimental studies.

Synergistic inhibition of Pseudomonas aeruginosa by EGCG and I3A: preliminary mechanisms and application in fish meat preservation.

Synergistic inhibition of Pseudomonas aeruginosa by EGCG and I3A: preliminary mechanisms and application in fish meat preservation.

Green synthesis of silver-modified bacterial cellulose with enhanced antimicrobial activity for advanced biomedical application.

Green synthesis of silver-modified bacterial cellulose with enhanced antimicrobial activity for advanced biomedical application.

Self-Adaptive Release of Stem Cell-Derived Exosomes from a Multifunctional Hydrogel for Accelerating MRSA-Infected Diabetic Wound Repair.

Chronic diabetic wounds are prone to severe skin necrosis and bacterial infections, with elevated reactive oxygen species (ROS) and persistent inflammation further hindering the healing process. Developing smart dressings with multifunctional therapeutic capabilities to simultaneously combat infections, reduce oxidative stress, alleviate inflammation, and promote tissue regeneration remains a significant challenge. Here, we introduce a self-adaptive yet multifunctional hydrogel (Exo-Gel) designed to accelerate methicillin-resistant Staphylococcus aureus (MRSA)-infected diabetic wound repair. Exo-Gel utilizes choline phosphate (CP) groups to both anchor stem cell-derived exosomes (Exo) via electrostatic interactions and disrupt bacterial membranes, providing inherent bacteriostatic effects. Additionally, ROS-responsive thioketal (TK) linkers enable the self-adaptive release of exosomes based on local ROS levels while also scavenging excess ROS. This synergistic system facilitates wound healing by modulating oxidative stress, reducing inflammation, promoting M2 macrophage polarization, and enhancing cell proliferation, myofibroblast migration, angiogenesis, and collagen deposition to accelerate tissue regeneration. In diabetic Sprague-Dawley rats with MRSA-infected full-thickness wounds, Exo-Gel achieved remarkable bacteriostatic activity and accelerated wound healing. Exo-Gel offers a cost-effective, multifunctional, and self-adaptive therapeutic strategy for managing chronic diabetic wounds, requiring no external components or operations, making it highly practical and translatable for clinical applications.

Individual and Simultaneous Photodegradation of Trimethoprim and Sulfamethoxazole Assessed with the Microbial Assay for Risk Assessment.

Co-trimoxazole is an antibacterial drug, a mixture of sulfamethoxazole (SMX) and trimethoprim (TMP) in a ratio of 5:1. Due to the different susceptibility of both components to decomposition under the influence of sunlight, the aim of the study was to assess the change in bacteriostatic activity during irradiation of the mixture of these antibiotics in a sunlight simulator. The bacteriostatic activity was assessed using a Microbial Assay for Risk Assessment (MARA), monitoring drug concentrations as well as the formation of photodegradation products using liquid chromatography (LC). The toxicity analysis of the SMX and TMP mixture showed synergistic bacteriostatic activity for six bacterial strains. This activity was maintained even during sample irradiation when 80-90% of SMX was degraded. This may indicate the bacteriostatic effect of SMX photoproducts and/or the lack of necessity to maintain a 5:1 ratio between SMX and TMP to maintain a strong effect of the mixture of these antibiotics. Analysis using LC with a high-resolution mass spectrometry detector revealed the presence of 11 SMX degradation products, including two with preserved sulfonamide structure, which may exhibit bacteriostatic activity.

Synthesis, Evaluation of Biological Activity, and Structure-Activity Relationships of New Amidrazone Derivatives Containing Cyclohex-1-ene-1-Carboxylic Acid.

In recent years, the incidence of acute and chronic inflammatory diseases has increased significantly worldwide, intensifying the search for new therapeutic agents, especially anti-inflammatory drugs. Therefore, the aim of this work was to synthesize, biologically assess, and explore the structure-activity relationships of new compounds containing the cyclohex-1-ene-1-carboxylic acid moiety. Six new derivatives, 2a-2f, were synthesized through the reaction of amidrazones 1a-1f with 3,4,5,6-tetrahydrophthalic anhydride. Their toxicity was evaluated in cultures of human peripheral blood mononuclear cells (PBMCs). Additionally, their antiproliferative properties and effects on the synthesis of TNF-α, IL-6, IL-10, and IL-1β were assessed in mitogen-stimulated PBMCs. The antimicrobial activity of derivatives 2a-2f was determined by measuring the minimal inhibitory concentration (MIC) values against five bacterial strains-Staphylococcus aureus, Mycobacterium smegmatis, Escherichia coli, Yersinia enterocolitica, and Klebsiella pneumoniae-and the fungal strain Candida albicans. All compounds demonstrated antiproliferative activity, with derivatives 2a, 2d, and 2f at a concentration of 100 µg/mL being more effective than ibuprofen. Compound 2f strongly inhibited the secretion of TNF-α by approximately 66-81% at all studied doses (10, 50, and 100 µg/mL). Derivative 2b significantly reduced the release of cytokines, including TNF-α, IL-6, and IL-10, at a high dose (by approximately 92-99%). Compound 2c exhibited bacteriostatic activity against S. aureus and M. smegmatis, while derivative 2b selectively inhibited the growth of Y. enterocolitica (MIC = 64 µg/mL). Some structure-activity relationships were established for the studied compounds.

Design and Validation of a Simplified Method to Determine Minimum Bactericidal Concentration in Nontuberculous Mycobacteria.

Nontuberculous mycobacteria (NTM) infections are rising, particularly those by Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MAB). Treating NTM infections is challenging due to their poor response to antibiotics. This study aimed to optimize the treatment of NTM infection by selecting antibiotics with bactericidal activity for combination therapy. To do this, we used the minimum bactericidal concentration (MBC) determination approach to define bactericidal or bacteriostatic activity. We developed three main objectives: validate a new method to determine MBC based on a reincubation method, determine MBC values of 229 NTM clinical isolates using the reincubation method, and evaluate antibiotic stability in preincubated microtiter plates. First, we assessed the stability of the antibiotics included in SLOWMYCOI Sensititre™ microtiter plates. Five strains of MAC were studied comparing the minimum inhibitory concentrations (MICs) of those preincubated for seven days vs. non-incubated plates. Then, we evaluated the percentage of reproducibility of MBC values using two methods, reincubation and subculturing (standard or traditional method) in 30 MAC isolates. Finally, we validated the reincubation method and prospectively determined the MBC values of the 229 NTM clinical strains. Antibiotic stability: The MIC was equivalent after 7 and 14 days for all the antibiotics, except rifampicin, for which the MIC increased by 2- to 3-fold after preincubation. Reincubation method: The percentage of reproducibility of the MBC values between the two methods was 95.2% (range 76.6% to 100%). Prospective validation: MBC/MIC ratios revealed differential bactericidal activity for most antibiotics according to the different species, being bactericidal in M. avium and Mycobacterium xenopi, and predominantly bacteriostatic in MAB. Preincubation of Sensititre™ microtiter plates did not alter the MIC values of the antibiotics included except for rifampicin, suggesting a loss of activity. MBC determination can be easily performed by the Reincubation method presented. MBC values provide useful additional information regarding MIC values since the MBC/MIC ratio reveals whether antibiotics have bactericidal or bacteriostatic activity according to the species, which is pivotal for selecting the most adequate antibiotic combination to ensure efficient treatment management.

Discovery, Biological Activity, and Biosynthesis of Pinocicolin A, an Antibiotic Isocyanide Metabolite Produced by Penicillium pinophilum.

We discovered new natural products pinocicolins A (1) and B (2) from Penicillium pinophilum and determined the stereochemistry of these compounds by degradation and derivatization. Compound 1, containing two isocyanide groups, exhibited bacteriostatic activity against Gram-positive bacteria depending upon its copper-chelating activity. The biosynthesis of compound 1 was reconstructed in Aspergillus nidulans by heterologous expression of multidomain isocyanide synthase-nonribosomal peptide synthetase Pp_crmA and efflux pump Pp_crmE. This is the first report of the heterologous production of a fungal diisocyanide natural product, highlighting the multistep catalysis of CrmA family enzymes.

Cholesterol-Rich Antibiotic-Loaded Liposomes as Efficient Antimicrobial Therapeutics.

Liposomal antibiotics have demonstrated higher bacteriostatic and bactericidal activities than free drugs. In this study, we investigated the effects of cholesterol (Chol) content of liposomes, liposome concentration, and surface coating with polyethylene glycol (PEG) on the antimicrobial activity of moxifloxacin (MOX) liposomes against Staphylococcus epidermidis (ATCC 35984) (S.e). MOX-liposome compositions with increasing Chol content were evaluated for their susceptibility to planktonic S.e (growth inhibition, killing, and live-dead staining), as well as against pre-formed biofilms (crystal violet, MTT assay, and confocal microscopy). The MOX-liposomes prepared by active loading were characterized in terms of loading, size distribution, and zeta potential. All liposomes had nano-dimensions ranging in diameter from 92nm to 114nm, with zeta-potential values from -2.30mV to -4.50mV. Planktonic bacteria and established biofilms are significantly more susceptible to MOX-liposomes with higher Chol-content than other liposome-types, and the same MOX dose encapsulated in 10 times higher lipids demonstrated higher antimicrobial activity. Coating the MOX liposomes with PEG did not affect their activity. Flow cytometry showed higher binding of Chol-rich liposomes to bacteria, explaining the higher antimicrobial activity. Interestingly, the integrity of calcein-loaded Chol-rich liposomes was much lower than that of liposomes with low or no Chol during incubation with various strains of S. epidermidis. In vivo results in a zebrafish infection model (bacteremia) confirmed the superior activity of Chol-rich MOX-liposomes compared to the free drug. The current in vitro and in vivo findings demonstrated the potential of PEGylated and Chol-rich liposomal antibiotics as highly efficient therapeutics for the treatment of S. epidermidis infections.

Optimizing ultrasonic extraction of polysaccharides from Spirulina platensis and evaluating their antioxidant and antibacterial activities in acidic environments.

Optimizing ultrasonic extraction of polysaccharides from Spirulina platensis and evaluating their antioxidant and antibacterial activities in acidic environments.

Functional analysis and modification of anti-lipopolysaccharide factor (ALF) from the freshwater crab Sinopotamon henanense and preparation of a novel ShALF6-2 K-AgNPs complex.

Functional analysis and modification of anti-lipopolysaccharide factor (ALF) from the freshwater crab Sinopotamon henanense and preparation of a novel ShALF6-2 K-AgNPs complex.

Extra peptidase of a cyanophage confers its stronger lytic effect on bloom-forming Microcystis aeruginosa.

Extra peptidase of a cyanophage confers its stronger lytic effect on bloom-forming Microcystis aeruginosa.

Mycoidesin, a novel lantibiotic, exhibits potent bacteriostatic activity against Listeria monocytogenes and effectively controls its growth in beef.

This study aimed to identify highly effective, stable, and safe natural bacteriocin preservatives with anti-Listeria monocytogenes activity. We isolated a novel class II lantibiotic, mycoidesin, which exhibited more efficient bacteriostatic activity against L. monocytogenes and increased stability compared to the applied bacteriocin food preservative, nisin A. Mycoidesin also showed favorable biosafety. Moreover, mycoidesin could be effectively used for controlling L. monocytogenes in beef, demonstrating its potential as a biopreservative to prevent L. monocytogenes-related contamination and improve the safety of meat and meat products in the agricultural and food industries.

In vitro Analysis of the Anticancer and Antidiabetic Effects of Teucrium orientale Leaf Hydrophilic Extract Grown in Two Palestinian Geographic Areas

Several studies have demonstrated that Teucrium orientale (T. orientale) species have therapeutic advantages, such as antioxidant, bacteriostatic, spasmolytic, and anti-inflammatory activity. This study aimed to assess the possible antidiabetic and anticancer activities of T. orientale leaf hydrophilic extracts collected from two distinct geographic regions in Palestine: Jerusalem and Ramallah. T. orientale hydrophilic extract was tested for its antidiabetic and anticancer properties on α-amylase activity and Lewis Lung Carcinoma (LLC) cells, respectively. The anticancer effect on LLC was evaluated by flow cytometry for cell proliferation and Annexin-V/propidium iodide (PI) staining for cell apoptosis. The T. orientale extract from Jerusalem had an IC50 of 7.43±0.84 μg/ml for inhibiting α-amylase enzyme activity, whereas the Ramallah extract had an IC50 value of 23.2±0.29 μg/ml. These values were compared to the positive control, Acarbose, which had an IC50 of 43.91±1.08 μg/ml. LLC cells were treated with one of the two extracts of T. orientale at different concentrations (0, 50, 100, 200, and 400 µg/ml) for 24 hours, and cell proliferation was assessed using an XTT assay. Total inhibition of LLC proliferation was achieved at 400 μg/ml in both extracts. The T. orientale extract from Jerusalem demonstrated a more efficient inhibitory effect at lower concentrations. Increasing concentrations of T. orientale (50, 100, 200, and 400 mg/ml) from the two geographic areas, Ramallah and Jerusalem, had no effect on the apoptosis rate in the control group. In contrast, elevated rates of apoptosis were observed following treatment with T. orientale extract in LLC cells at all tested concentrations, and this was positively associated with the late apoptosis marker Annexin-V+/PI+. Moreover, the T. orientale extract from Jerusalem exhibited an apoptotic rate of 90±3.4% at the highest concentration of 400 mg/ml, compared to 62.6±3.4% following treatment with the Ramallah extract. This suggests that the T. orientale extract from Jerusalem induced apoptosis in LLC cells more efficiently than the extract from Ramallah. The extracts derived from T. orientale show promising potential as a natural antidiabetic and anticancer agent, as evidenced by their ability to inhibit the α-amylase enzyme, impede the growth of LLC cells, and enhance apoptosis. Further in vivo and preclinical investigations are required to validate these effects.

Comprehensive Phenotypic Characterization and Genomic Analysis Unveil the Probiotic Potential of Bacillus velezensis K12.

Bacillus spp. have emerged as pivotal sources of probiotic preparations, garnering considerable attention in recent years owing to their vigorous bacteriostatic activity and antimicrobial resistance. This study aimed to investigate these probiotic characteristics in depth and verify the safety of Bacillus velezensis K12, a strain isolated from broiler intestine. The K12 strain was identified as Bacillus velezensis based on its morphology and 16S rDNA sequence homology analysis. Subsequently, B. velezensis K12 was evaluated for acid resistance, bile salt resistance, gastrointestinal tolerance, drug sensitivity, and antimicrobial activity. Additionally, whole-genome sequencing technology was employed to dissect its genomic components further, aiming to explore its potential applications as a probiotic strain. B. velezensis K12 was sensitive to six antibiotics and had acid tolerance. Furthermore, it showed potent antimicrobial activity against a wide range of pathogenic bacteria, including Escherichia coli (E. coli), Staphylococcus aureus, Salmonella, Clostridium perfringens, Bacillus cereus, and Vibrio parahaemolyticus. The complete genome sequencing of B. velezensis K12 revealed a genomic length of 3,973,105 base pairs containing 4123 coding genes, among which 3973 genes were functionally annotated. The genomic analysis identified genes associated with acid and bile tolerance, adhesion, antioxidants, and secondary metabolite production, whereas no functional genes related to enterotoxins or transferable antibiotic resistance were detected, thereby confirming the probiotic properties of B. velezensis K12. B. velezensis K12 exhibits broad-spectrum bacteriostatic activity and in vitro safety, positioning it as a potential candidate strain for developing probiotic Bacillus preparations.

From Tradition to Innovation: Pinellia ternata Extracts Stabilized Selenium Nanoparticles for Enhanced Bioactivity.

Selenium nanoparticles (SeNPs) are widely used for their effective antioxidative, anti-tumor, and antibacterial properties with minimal toxicity. Developing eco-friendly and simple synthesis methods for SeNPs with improved bioactivity, biocompatibility, and stability is crucial. This study synthesized bioactive SeNPs using extracts from the traditional Chinese medicine Pinellia ternata as a stabilizing agent. SeNPs were characterized to have an amorphous structure, spherical shape, uniform dispersion, and an average diameter of 54.6nm. They had a surface charge of - 28.2mV, enhancing stability through interactions with compounds in Pinellia ternata extracts. The synthesized SeNPs demonstrated strong antioxidant and antibacterial properties in vitro, outperforming Na2SeO3 and Pinellia ternata extracts. Specifically, SeNPs showed bacteriostatic activity against Escherichia coli (E. coli) at 120, 240, and 480µg/mL, with a lasting effect at 480µg/mL after 23h. Staphylococcus aureus (S. aureus) was more sensitive, with significant antimicrobial and bactericidal effects at 15, 30, and 60µg/mL, achieving complete inhibition at 60µg/mL after 20h. In zebrafish exposure experiments, SeNPs at low (0.2mg/L) and medium (0.5mg/L) concentrations enhanced antioxidant enzyme activities in the liver without inducing significant acute toxicity, whereas high concentrations (1mg/L) resulted in oxidative damage to the liver and gills. This study introduces a novel method for synthesizing a stable SeNP-herbal medicine extracts composite with outstanding biological activity and biosafety, potentially expanding the applications of herbal medicine beyond traditional antioxidant and antimicrobial treatments.

Bacteriostatic activity and mechanism of minerals containing rubidium

Abstract Background Metals and their ions have been used to reduce bacterial infection risks. Among them, minerals containing rubidium (MCR), natural minerals containing metal ions, show potential as novel and tunable materials. Objectives This study aimed to investigate the antibacterial activity and mechanism of MCR. Methods The inhibitory effect of MCR on bacteria was clarified using the growth curve method, turbidimetric method, and minimum inhibitory concentration method. Physiological and biochemical indices were employed to investigate the inhibitory mechanism of MCR. Results The results revealed that MCR inhibited Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli with minimum inhibitory concentrations of 11.95, 2.60, and 2.60 mg/mL, respectively. The inhibitory activity of MCR was insignificant against Bacillus subtilis, Salmonella typhimurium, and Helicobacter pylori at 3.25 mg/mL. Mechanistic assessments showed that MCR affected bacterial conductivity, protein and nucleic acid levels, reducing sugar content, respiratory chain dehydrogenase activity, bacterial lipid peroxidation, intracellular adenosine triphosphate, and extracellular alkaline phosphatase. Conclusions MCR has bacteriostatic activity and the mechanism primarily involves adhesion to bacteria, disrupting the integrity of their cell walls and membranes, and altering their permeability. This disruption leads to the release of intracellular molecules of various sizes, inhibiting cellular respiration and metabolism, and causing oxidative damage. These combined effects impair cellular functions, affecting cell growth and metabolism, or leading to cell death. These findings provide a theoretical reference for the development of MCR as a bacteriostatic agent.

Evaluation of Nonthermal Technologies to Reduce or Replace Nitrite in Meat Products.

Nitrite and nitrate salts are preservatives that act as antimicrobial (bacteriostatic and bactericidal activity) and antioxidant agents in the processing of meat products and confer sensory properties to meat (by creating and preserving colours and flavours). Nitrite is mainly used as a preservative to prevent the growth of Clostridium botulinum and the production of its toxins. However, nitrite and nitrate are also associated with the production of N-nitroso compounds, such as carcinogenic N-nitrosamines, which can have adverse health effects. Therefore, the health risks of these preservatives must be weighed against the need to prevent foodborne pathogens, especially spores of C. botulinum, from infecting food. In this review, we discuss the advantages and disadvantages of using nonthermal technologies as a strategy to partially or totally replace nitrite in meat products, particularly regarding antimicrobial efficacy and N-nitrosamine formation. Methods such as high-pressure processing, pulsed electric fields and cold plasma have been studied for these purposes, but these technologies can alter the sensory properties and stability of foods. Nevertheless, irradiation at lower doses has great potential as a tool for reformulation of cured meat products. It contributes to the reduction of the residual nitrite and consequently to the production of N-nitrosamines while ensuring microbiological safety without significant changes in the product quality.

Bacteriostatic Activity and Resistance Mechanism of Artemisia annua Extract Against Ralstonia solanacearum in Pepper.

The destructive bacterial wilt disease caused by Ralstonia solanacearum leads to substantial losses in pepper production worldwide. Plant-derived pesticides exhibit advantages of high efficiency and broad spectrum when compared to traditional chemical pesticides. Artemisia annua and 'Tai Jiao' No. 1 were used as the experimental materials, and treated with 0.75 g·mL-1, 1.5 g·mL-1, and 3 g·mL-1 of A. annua extract and inoculated with R. solanacearum at a concentration of OD600 = 0.1 for 14 days. The inhibitory activity of A. annua extracts against R. solanacearum, as well as the disease index, defense enzyme activities, and defense-related substances contents of pepper seedlings were determined. The results showed that the Minimum Inhibitory Concentration (MIC) of the A. annua extract was 3 g·mL-1. As the concentration of A. annua extract increased, the extent of R. solanacearum cell crumpling intensified, accompanied by a gradual decline in its biofilm-forming ability. On the 14th day after treatment, the disease severity index and incidence rate were significantly reduced when the A. annua extract was applied at concentrations of 0.75 g·mL-1 and 3 g·mL-1. At both the 7th and 14th days after treatment, the application of A. annua extract at concentrations of 0.75 g·mL-1 and 3 g·mL-1 led to enhanced activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in peppers at different stages. Simultaneously, it reduced the levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2), effectively scavenging reactive oxygen species and alleviating cellular lipid peroxidation. Furthermore, the extract increased the activities of polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL), as well as elevated the contents of soluble protein, flavonoids, and total phenols, ultimately enhancing the disease resistance of peppers. Considering the development costs, the application of A. annua extract at a concentration of 0.75 g·mL-1 demonstrates great potential for green control measures against bacterial wilt in peppers.

Ruminal fermentation and in vitro digestibility of beef cattle fed monensin and monensin and virginiamycin or monensin and mineral traces + yeast combination in high-grains diets

This work was realized to study the antimicrobial activity, pH, ammonia, antioxidant activity, volatile acid fatty and in the liquor rumen of different additives combinations (monensin – MONE, monensin + virginiamycin – MO+VI and monensin + mineral traces + yeast – MO+MY) compared to control – CONT (without additives). The antimicrobial activity was studied against four strains of Gram-positives ruminal bacteria and seven strains of Gram-negatives ruminal bacteria. The concentration of MONE was 37.7 g/L, MO+VI was 42.7 g/L and MO+ML was 39.3 g/L. Cultivation was performed at 39° C, and bacteria growth was evaluated by quantifying the optical density (OD) at 600 nm using a spectrophotometer. Optical density was evaluated before and 8, 12, and 24 hours after incubation. The Ruminococcus albus, Ruminococcus flavefaciens and Lachnospira multipara bacteria growth was inhibited (P < 0.05) by MONE, MO+VI and MO+MY treatments at the three times (8, 12 and 24 hours). The greatest growth inhibition of these three bacteria occurred in the MO+VI treatment, in comparison with the MONE only or the MO+MY combinations. MONE e MO+MY treatments showed a sign of bacteriostatic activity; whereas the MO+VI treatment indicates a sign of bactericidal action. The three compounds studied showed inhibitory activity lower against Streptococcus bovis growth in the three studied periods (8, 12 and 24 hours). The greatest inhibitory activity was observed in the MO+VI additives’ combinations and the lowest for the MO+MY additives combinations, being intermediate for monensin only. The additives addition had a little effect on the microbial growth of Prevotella albensis and Prevotella bryantii bacteria, intermediary on Prevotella ruminicola and Anaerovibrio lipolyticus bacteria and strong activity on Ruminobacter amylophilus.