Antimicrobial effect of different types of honey on Staphylococcus aureus

Honey exhibits antimicrobial activities against a wide range of bacteria in different milieu. This study aims to compare the effects of five types of honey (both imported and local Saudi honey) against Staphylococcus aureus. The five types of honey (Manuka Honey UMF +20, Manuka Honey UMF +16, Active +10 Manuka Honey, Sidr honey and Nigella sativa honey) were evaluated for their bactericidal/bacteriostatic activities against both methicillin resistant and sensitive S. aureus. The inhibitory effect of honey on bacterial growth was evident at concentrations of 20% and 10% (v/v). Manuka Honey showed the best results. Manuka Honey UMF +20 had a bactericidal effect on both methicillin resistant and sensitive S. aureus. However, Sidr and N. sativa honey exerted only a bacteriostatic effect. The efficacy of different types of honey against S. aureus was dependent on the type of honey and the concentration at which it was administered. Manuka Honey had the best bactericidal activity. Future experiments should be conducted to evaluate the effects of honey on bacterial resistance.     

Potential effects of Saudi Shaoka (Fagonia bruguieri) honey against multi-drug-resistant bacteria and cancer cells in comparison to Manuka honey

The global spread of antimicrobial-resistant infectious diseases and cancer are the most widespread public health issue and has led to high mortality rates. This study aims to evaluate and verify the antibacterial and antitumor activities of Shaoka and Manuka honey against pathogenic bacteria, human hepatocarcinoma (HepG2) and breast cancer (MCF-7) cell lines. Shaoka hone was analyzed using HPLC, UV–vis, and GC/MC, while antibacterial activity was measured by agar diffusion, broth microdilution methods, and Transmission Electron Microscopy (TEM). Antitumor activity was investigated morphologically and by MTT assay. According to the presented data of HPLC analysis, Shaoka honey was generally richer in polyphenolic components, the antibacterial activity showed that Shaoka honey is equivalent or relatively more active than Manuka honey against a broad spectrum of multi-drug-resistant bacteria. It inhibited the growth of ESBL Escherichia coli in the absence or presence of catalase enzyme with a concentration approximately 8.5%–7.3% equivalent to phenol, which supported the highest level of non-peroxide-dependent activity. The minimum bactericidal concentrations (MBCs) ranged between 5.0% and 15.0% honey (w/v). TEM observation revealed distorted cell morphology, cytoplasmic shrinkage, and cell wall destruction of treated bacteria. The selected honey exerted cytotoxicity on both cancer cell lines, inhibiting cell proliferation rate and viability percent in HepG2 and MCF-7 cancer cells, by different degrees depending on the honey quality, Shaoka honey competed Manuka inhibitory effects against both cancer cells. The obtained data confirmed the potential for use of Saudi Shaoka honey as a remedy, this well introduces a new honey template as medical-grade honey for treating infectious disease and cancer.     

Identification and characterization of an anti-pseudomonal dichlorocarbazol derivative displaying anti-biofilm activity

Pseudomonas aeruginosa strains resistant towards all currently available antibiotics are increasingly encountered, raising the need for new anti-pseudomonal drugs. We therefore conducted a medium-throughput screen of a small-molecule collection resulting in the identification of the N-alkylated 3,6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (MIC = 18.5 μg mL⁻¹). This compound, compound 1, is bacteriostatic towards a broad spectrum of Gram-positive and Gram-negative pathogens, including P. aeruginosa. Importantly, 1 also eradicates mature biofilms of P. aeruginosa. 1 displays no cytotoxicity against various human cell types, pointing to its potential for further development as a novel antibacterial drug.     

Pseudomonas aeruginosa isolates of distinct sub-genotypes exhibit similar potential of antimicrobial resistance by drugs exposure

Pseudomonas aeruginosa, a wide-spread opportunistic pathogen, often complicates clinical treatments due to its resistance to a large variety of antimicrobials, especially in immune compromised patients, occasionally leading to death. However, the resistance to antimicrobials varies greatly among the P. aeruginosa isolates, which raises a question on whether some sub-lineages of P. aeruginosa might have greater potential to develop antimicrobial resistance than others. To explore this question, we divided 160 P. aeruginosa isolates collected from cities of USA and China into distinct genotypes using I-CeuI, a special endonuclease that had previously been proven to reveal phylogenetic relationships among bacteria reliably due to the highly conserved 26-bp recognition sequence. We resolved 10 genotypes by I-CeuI analysis and further divided them into 82 sub-genotypes by endonuclease cleavage with SpeI. Eight of the 10 genotypes contained both multi-drug resistant (MDR) and less resistant isolates based on comparisons of their antimicrobial resistance profiles (ARPs). When the less resistant or susceptible isolates from different genotypes were exposed to eight individual antimicrobials, they showed similar potential to become resistant with minor exceptions. This is to our knowledge the first report to examine correlations between phylogenetic sub-lineages of P. aeruginosa and their potential to become resistant to antimicrobials. This study further alerts the importance and urgency of antimicrobial abuse control.     

Resistance to Rhizoctonia solani and Presence of Antimicrobial Compounds in Camelina sativa Roots

Camelina sativa (L.) Crantz was significantly more resistant to Rhizoctonia solani Kühn than [itBrassica napus L. cv Westar. Emergence of C. sativa seedlings was 22 to 33% greater than those of Westar in R. solani-infested soil. The greater resistance of C. sativa seedlings to R. solani appeared to be due to greater amounts of antimicrobial compounds present in C. sativa roots. These antimicrobial compounds inhibited the growth of both weakly virulent and virulent R. solani] isolates to the same extent. Four antimicrobial compounds were purified from C. sativa roots and their structures elucidated. Two were identified as the phytoalexins (camalexin and methoxycamalexin) previously described from C. sativa leaves. This appears to be the first report of elicitation of phytoalexins from roots of crucifers. Two preformed antimicrobial compounds were identified as methyl 1-methylindole-3-carboxylate and 10-methyl sulfinyldecylisothiocyanate.     

In vitro antimicrobial activity against Pseudomonas aeruginosa and acute oral toxicity of marine algae Gracilaria changii

Methanol extract of the Gracilaria changii has been screened for antimicrobial activity against Pseudomonas aeruginosa. Antimicrobial activities were carried out using disc diffusion assay and broth dilution method against P. aeruginosa. The methanol extract of G. changii showed a good antimicrobial activity against P. aeruginosa with MIC (Minimum Inhibitory Concentration) value of 6.25 mg/ml. Exposure of P. aeruginosa cells to 6.25 mg/ml of methanol extract of G. changii resulted in complete inhibition of the bacterial cells. The main abnormalities noted via SEM and TEM studies were the alterations in morphology and cytology of the bacterial cells. The main reason for this deterioration was discussed. The effect of the methanol extract on the growth profile for the bacteria was also done and confirmed the bactericidal effect of the G. changii methanol extract on P. aeruginosa by changing the normal growth profile of P. aeruginosa. In an acute toxicity study using mice, the median lethal dose (LD₅₀) of the extract was greater than 2000 mg/kg, and we found no pathological changes in macroscopic examination by necropsy of mice treated with extract. We conclude that G. changii might be safely used as an antimicrobial agent.     

Synthesis and structural characterization of silver nanoparticles using bacterial exopolysaccharide and its antimicrobial activity against food and multidrug resistant pathogens

A green, simple, and effective approach was performed to synthesize potent silver nanoparticles (SNPs) using bacterial exopolysaccharide as both a reducing and stabilizing agent. The synthesized SNPs were characterized using UV-vis spectroscopy, transmission electron microscopy, energy dispersive X-ray analysis, X-ray diffraction, and Fourier-transform-infrared spectra analyses. The SNPs varied in shape and were multidispersed with a mean diameter of 10 nm ranging from 2 to 15 nm and were stable up to 2 months at room temperature. The antimicrobial activity of the SNPs was analyzed against bacterial and fungal pathogens using the agar well diffusion method. Dose dependent inhibition was observed for all bacterial pathogens. The multidrug resistant pathogens P. aeruginosa and K. pneumonia were found to be more susceptible to the SNPs than the food borne pathogen L. monocytogenes. The fungi Aspergillus spp. exhibited a maximum zone of inhibition compared to that of Penicillum spp. These results suggest that exopolysaccharide-stabilized SNPs can be used as an antimicrobial agent for various biomedical applications.     

Antimicrobial activity of some plant extracts against bacterial strains causing food poisoning diseases

Prevention of food spoilage and food poisoning pathogens is usually achieved by use of chemical preservatives which have negative impacts including: human health hazards of the chemical applications, chemical residues in food & feed chains and acquisition of microbial resistance to the used chemicals. Because of such concerns, the necessity to find a potentially effective, healthy safer and natural alternative preservatives is increased. Within these texts, Plant extracts have been used to control food poisoning diseases and preserve foodstuff. Antimicrobial activity of five plant extracts were investigated against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi using agar disc diffusion technique. Ethanolic extracts of Punica granatum, Syzygium aromaticum, Zingiber officinales and Thymus vulgaris were potentially effective with variable efficiency against the tested bacterial strains at concentration of 10 mg/ml while extract of Cuminum cyminum was only effective against S. aureus respectively. P. granatum and S. aromaticum ethanolic extracts were the most effective plant extracts and showed bacteriostatic and bactericidal activities against the highly susceptible strains of food borne pathogenic bacteria (S. aureus and P. aeruginosa) with MIC's ranged from 2.5 to 5.0 mg/ml and MBC of 5.0 and 10 mg/ml except P. aeruginosa which was less sensitive and its MBC reached to 12.5 mg/ml of S. aromaticum respectively. These plant extracts which proved to be potentially effective can be used as natural alternative preventives to control food poisoning diseases and preserve food stuff avoiding healthy hazards of chemically antimicrobial agent applications.     

Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens

The antimicrobial properties of 21 plant essential oils and two essences were investigated against five important food-borne pathogens, Campylobacter jejuni, Salmonella enteritidis, Escherichia coli, Staphylococcus aureus and Listeria monocytogenes. The oils of bay, cinnamon, clove and thyme were the most inhibitory, each having a bacteriostatic concentration of 0·075% or less against all five pathogens. In general, Gram-positive bacteria were more sensitive to inhibition by plant essential oils than the Gram-negative bacteria. Campylobacter jejuni was the most resistant of the bacteria investigated to plant essential oils, with only the oils of bay and thyme having a bacteriocidal concentration of less than 1%. At 35 °C, L. monocytogenes was extremely sensitive to the oil of nutmeg. A concentration of less than 0·01% was bacteriostatic and 0·05% was bacteriocidal, but when the temperature was reduced to 4 °C, the bacteriostatic concentration was increased to 0·5% and the bacteriocidal concentration to greater than 1%.     

In vitro activity of some natural honeys against Entamoeba histolytica and Giardia lamblia trophozoites

Considering the potentiality of honey in combating diseases, the present study was carried out aiming to assess the in vitro antiprotozoal activity of several honeys (Ziziphus spina-christi, Acacia nilotica, Acacia seyal, and Cucurbita maxima) against Entamoeba histolytica and Giardia lamblia by employing the sub-culture method. All the tested honeys inhibited the growth of trophozoites, and the level of inhibition varied according to the assayed concentrations and incubation times. Acacia seyal honey had completely stopped motility of E. histolytica trophozoites at a concentration ≤ 50 µg/ml after incubation for 72 h. Ziziphus spina-christi, Acacia seyal, and Acacia nilotica honeys had completely inhibited the growth of Giardia lamblia trophozoites at concentration ≤ 200 µg/ml after 72 h. These inhibitory activities were similar to that of Metronidazole? which showed IC₅₀ = 0.27. The mammalian cytotoxicity of these honeys against normal Vero cell line which determined by applying MTT method verified the nontoxicity of the examined honeys. Also the proximate composition of the samples indicated compliance with the natural honey standards. The findings of the study indicate the need for in vivo studies and further investigations to identify active principles with antiprotozoal activities from natural honeys.     

Synergistic interactions in two-drug and three-drug combinations (thymol,EDTA and vancomycin) against multi drug resistant bacteria including E. coli

Combinations of two or more drugs, which affect different targets, have frequently been used as a new approach against resistant bacteria. In our work we studied the antimicrobial activity (MIC, MBC) of individual drugs (the phenolic monoterpene thymol, EDTA and vancomycin), of two-drug interactions between thymol and EDTA in comparison with three-drug interactions with vancomycin against sensitive and resistant bacteria. Thymol demonstrated moderate bactericidal activity (MBC between 60 and 4000 μg/ml) while EDTA only exhibited bacteriostatic activity over a range of 60–4000 μg/ml. MICs of vancomycin were between 0.125 and 16 μg/ml against Gram-positive and between 32 and 128 μg/ml against Gram-negative bacteria. Checkerboard dilution and time-kill curve assays were performed to evaluate the mode of interaction of several combinations against Methicillin-resistant Staphylococcus aureus (MRSA NCTC 10442) and Escherichia coli (ATCC 25922). Checkerboard data indicate indifferent interaction against Gram-positive (FICI = 1–1.3) and synergy against Gram-negative bacteria (FICI ≈ 0.4), while time kill analyses suggest synergistic effect in different combinations against both types of bacteria. It is remarkable that the combinations could enhance the sensitivity of E. coli to vancomycin 16-fold to which it is normally insensitive. We have provided proof for the concept, that combinations of known antibiotics with modern phytotherapeutics can expand the spectrum of useful therapeutics.     

Investigation of the antimicrobial activity of soy peptides by developing a high throughput drug screening assay

BackgroundAntimicrobial resistance is a great concern in the medical community, as well as food industry. Soy peptides were tested against bacterial biofilms for their antimicrobial activity. A high throughput drug screening assay was developed using microfluidic technology, RAMAN spectroscopy, and optical microscopy for rapid screening of antimicrobials and rapid identification of pathogens.MethodsSynthesized PGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides were tested against Pseudomonas aeruginosa and Listeria monocytogenes using a microdilution assay. Microfluidic technology in combination with Surface Enhanced RAMAN Spectroscopy (SERS) and optical microscopy was used for rapid screening of soy peptides, pathogen identification, and to visualize the impact of selected peptides.ResultsThe PGTAVFK peptide did not significantly affect P. aeruginosa, although it had an inhibitory effect on L. monocytogenes above a concentration of 625 µM. IKAFKEATKVDKVVVLWTA was effective against both P. aeruginosa and L. monocytogenes above a concentration of 37.2 µM. High throughput drug screening assays were able to reduce the screening and bacterial detection time to 4 h. SERS spectra was used to distinguish the two bacterial species.ConclusionsPGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides showed antimicrobial activity against P. aeruginosa and L. monocytogenes. Development of high throughput assays could streamline the drug screening and bacterial detection process.General significanceThe results of this study show that the antimicrobial properties, biocompatibility, and biodegradability of soy peptides could possibly make them an alternative to the ineffective antimicrobials and antibiotics currently used in the food and medical fields. High throughput drug screening assays could help hasten pre-clinical trials in the medical field.     

The Effect of New Zealand Kanuka,Manuka and Clover Honeys on Bacterial Growth Dynamics and Cellular Morphology Varies According to the Species

Treatment of chronic wounds is becoming increasingly difficult due to antibiotic resistance. Complex natural products with antimicrobial activity, such as honey, are now under the spotlight as alternative treatments to antibiotics. Several studies have shown honey to have broad-spectrum antibacterial activity at concentrations present in honey dressings, and resistance to honey has not been attainable in the laboratory. However not all honeys are the same and few studies have used honey that is well defined both in geographic and chemical terms. Here we have used a range of concentrations of clover honey and a suite of manuka and kanuka honeys from known geographical locations, and for which the floral source and concentration of methylglyoxal and hydrogen peroxide potential were defined, to determine their effect on growth and cellular morphology of four bacteria: Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. While the general trend in effectiveness of growth inhibition was manuka>manuka-kanuka blend>kanuka>clover, the honeys had varying and diverse effects on the growth and cellular morphology of each bacterium, and each organism had a unique response profile to these honeys. P. aeruginosa showed a markedly different pattern of growth inhibition to the other three organisms when treated with sub-inhibitory concentrations of honey, being equally sensitive to all honeys, including clover, and the least sensitive to honey overall. While hydrogen peroxide potential contributed to the antibacterial activity of the manuka and kanuka honeys, it was never essential for complete growth inhibition. Cell morphology analysis also showed a varied and diverse set of responses to the honeys that included cell length changes, cell lysis, and alterations to DNA appearance. These changes are likely to reflect the different regulatory circuits of the organisms that are activated by the stress of honey treatment.     

Synthesis and antimicrobial evaluation of cationic low molecular weight amphipathic 1,2,3-triazoles

A library of 28 small cationic 1,4-substituted 1,2,3-triazoles was prepared for studies of antimicrobial activity. The structures addressed the pharmacophore model of small antimicrobial peptides and an amphipathic motif found in marine antimicrobials. Eight compounds showed promising antimicrobial activity, of which the most potent compound 10b displayed minimum inhibitory concentrations of 4–8 μg/mL against Streptococcus agalacticae, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis. The simple syntheses and low degree of functionalization make these 1,4-substituted 1,2,3-triazoles interesting for further optimizations.     

The origin of methylglyoxal in New Zealand manuka (Leptospermum scoparium) honey

Methylglyoxal in New Zealand manuka honey has been shown to originate from dihydroxyacetone, which is present in the nectar of manuka flowers in varying amounts. Manuka honey, which was freshly produced by bees, contained low levels of methylglyoxal and high levels of dihydroxyacetone. Storage of these honeys at 37 °C led to a decrease in the dihydroxyacetone content and a related increase in methylglyoxal. Addition of dihydroxyacetone to clover honey followed by incubation resulted in methylglyoxal levels similar to those found in manuka honey. Nectar washed from manuka flowers contained high levels of dihydroxyacetone and no detectable methylglyoxal.     

Antibiotic and synergistic effect of Leu-Lys rich peptide against antibiotic resistant microorganisms isolated from patients with cholelithiasis

Pseudomonas aeruginosa has eventually developed resistance against flomoxef sodium, isepamicin and cefpiramide. Therefore, in this study, the antibacterial activity and synergistic effects of the amphipathic-derived P5-18mer antimicrobial peptide were tested against pathogens associated with cholelithiasis that have developed resistance against commonly used antibiotics. The results were then compared with the activities of the amphipathic-derived peptide, P5-18mer, melittin and common antibiotics. Growth inhibition of planktonic bacteria was tested using the National Committee for Clinical Laboratory Standards (NCCLS). The bactericidal activity of the antimicrobial peptides was measured using time-kill curves. Synergistic effects were evaluated by testing the effects of P5-18mer alone and in combination with flomoxef sodium, isepamicin or cefpiramide at 0.5 × MIC. P5-18mer peptide displayed strong activity against pathogens and flomoxef sodium, isepamicin and cefpiramide-resistant bacteria cell lines obtained from a patient with gallstones; however, it did not exert cytotoxicity against the human keratinocyte HaCat cell line. In addition, the results of time-kill curves indicated that P5-18mer peptide exerted bactericidal activity against four strains of P. aeruginosa. Finally, the use of P5-18mer and antibiotics exerted synergistic effects against cell lines that were resistant to commonly used antibiotics. These results indicate that this class of peptides has a rapid microbicidal effect on flomoxef sodium, isepamicin and cefpiramide-resistant strains of P. aeruginosa. Therefore, these peptides may be used as a lead drug for the treatment of acquired pathogens from patients with cholelithiasis who are affected with antibiotic-resistant bacteria.     

Antibiofilm properties of chemically synthesized silver nanoparticles found against Pseudomonas aeruginosa

Nanomedicine is now being introduced as a recent trend in the field of medicine. It has been documented that metal nanoparticles have antimicrobial effects for bacteria, fungi and viruses. Recent advances in technology has revived the use of silver nanoparticles in the medical field; treatment, diagnosis, monitoring and control of disease. It has been used since ancient times for treating wide range of illnesses. Bacterial cells adheres to surfaces and develop structures known as biofilms. These structures are natural survival strategy of the bacteria to invade the host. They are more tolerant to commonly used antimicrobial agents, thus being more difficult to be controlled. This leads to increase in severity of infection. In this study, we have investigated the effect of silver nanoparticles in the formation of biofilm in multidrug resistant strains of Pseudomonas aeruginosa. Observation showed that biofilm formation occurred at bacterial concentration of 10⁶ cfu/ml for the sensitive strain of P. aeruginosa while in the resistant strain, the biofilm was evident at bacterial concentration of about 10³ cfu/ml. The biofilm were then tested against various concentrations of silver nanoparticles to determine the inhibitory effect of the silver nanoparticles. In the sensitive strain, 20 μg/ml of silver nanoparticles inhibited the growth optimally at bacterial concentration of 10⁴ cfu/ml with an inhibition rate of 67%. Similarly, silver nanoparticles inhibited the formation of biofilm in the resistant strain at an optimal bacterial concentration of 10⁵ cfu/ml with an inhibition rate of 56%. Thus, silver nanoparticles could be used as a potential alternative therapy to reduce severity of disease due to P. aeruginosa infections.     

Synergism between Medihoney and Rifampicin against Methicillin-Resistant Staphylococcus aureus (MRSA)

Skin and chronic wound infections caused by highly antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are an increasing and urgent health problem worldwide, particularly with sharp increases in obesity and diabetes. New Zealand manuka honey has potent broad-spectrum antimicrobial activity, has been shown to inhibit the growth of MRSA strains, and bacteria resistant to this honey have not been obtainable in the laboratory. Combinational treatment of chronic wounds with manuka honey and common antibiotics may offer a wide range of advantages including synergistic enhancement of the antibacterial activity, reduction of the effective dose of the antibiotic, and reduction of the risk of antibiotic resistance. The aim of this study was to investigate the effect of Medihoney in combination with the widely used antibiotic rifampicin on S. aureus. Using checkerboard microdilution assays, time-kill curve experiments and agar diffusion assays, we show a synergism between Medihoney and rifampicin against MRSA and clinical isolates of S. aureus. Furthermore, the Medihoney/rifampicin combination stopped the appearance of rifampicin-resistant S. aureus in vitro. Methylglyoxal (MGO), believed to be the major antibacterial compound in manuka honey, did not act synergistically with rifampicin and is therefore not the sole factor responsible for the synergistic effect of manuka honey with rifampicin. Our findings support the idea that a combination of honey and antibiotics may be an effective new antimicrobial therapy for chronic wound infections.     

Functional whole-colony screening method to identify antimicrobial peptides

A high throughput method for screening cDNA libraries has been developed to identify putative antimicrobial peptides (AMPs). It is based on a rapid dye inclusion assay for assessing antagonism of bacterial viability. Colonies are grown on a membrane on a permissive medium until full colony size is reached. The membrane, supporting the array of colonies, is transferred onto an inductive medium containing a vital dye. Upon expression of any antagonizing peptides, the cell membrane becomes compromised allowing dye infusion to permit visual identification of deleterious peptides.Our approach was validated by screening a synthetic oligonucleotide library expressed in Escherichia coli. A random oligonucleotide library, containing inserts of up to 75 nucleotides in length was constructed and expressed in E. coli. From a potential pool of 100 000 peptides, in a single round of screening, three were found to be antimicrobial: L1, L3, and L8. Peptide L1 was shown to have a concentration-dependent bactericidal effect against Gram-negative E. coli and moderate biostatic activity against the Gram-positive bacteria Listeria monocytogenes. L8 was found to have bacteriostatic, and possibly bactericidal effect against E. coli, Pseudomonas aeruginosa and Salmonella typhimurium. These results validated this high throughput AMP identification assay based on filter bound colony array libraries and vital dye inclusion.     

Lactobacillus plantarum isolated from molasses produces bacteriocins active against Gram-negative bacteria

Two bacteriocins, ST28MS and ST26MS, produced by Lactobacillus plantarum isolated from molasses, inhibited the growth of Lactobacillus casei, Lactobacillus sakei, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli and Acinetobacter baumanii. The mode of activity of the bacteriocins is bacteriostatic, as observed against L. casei and P. aeruginosa. Reduction in antimicrobial activity was recorded after treatment with Proteinase K, papain, trypsin, chymotrypsin, pronase, pepsin and protease. Both peptides remained active after 20 min at 121 °C. Bacteriocin ST28MS was produced at much higher levels (12,800 AU/mL) compared to bacteriocin ST26MS (6400 AU/mL) with glucose as carbon source. The activity of bacteriocin ST28MS decreased by 50% at pH below 4.0. Bacteriocin ST26MS, on the other hand, is more stable at this pH. Production of both bacteriocins is stimulated by tryptone. Potassium (KH₂PO₄ and K₂HPO₄) at 5 and 10 g/L stimulated the production of bacteriocin ST28MS, but not bacteriocin ST26MS. MRS supplemented with glycerol (1–5 g/L) did not result in any changes in the activity levels of the two bacteriocins. Ascorbic acid and Vitamins B₁ and B₁₂ are required for bacteriocin ST28MS production, but only Vitamin B₁₂ for bacteriocin ST26MS production. No plasmids were recorded for strains ST28MS and ST26MS, suggesting that the genes encoding production of the two bacteriocins are located on the genomes.