Antimicrobial Susceptibility of Persister Biofilm Cells of Bacillus cereus and Pseudomonas fluorescens

The present study evaluates the antimicrobial susceptibility of persister cells of Bacillus cereus and Pseudomonas fluorescens after their regrowth in suspension and as biofilms. Two conventional (benzalkonium chloride—BAC and peracetic acid—PAA) and two emerging biocides (glycolic acid—GA and glyoxal—GO) were selected for this study. Persister cells resulted from biofilms subjected to a critical treatment using the selected biocides. All biocide treatments developed B. cereus persister cells, except PAA that effectively reduced the levels of vegetative cells and endospores. P. fluorescens persister cells comprise viable and viable but non-culturable cells. Afterwards, persister cells were regrown in suspension and in biofilms and were subjected to a second biocide treatment. In general, planktonic cultures of regrown persister cells in suspension lost their antimicrobial tolerance, for both bacteria. Regrown biofilms of persister cells had antimicrobial susceptibility close to those regrown biofilms of biocide-untreated cells, except for regrown biofilms of persister P. fluorescens after BAC treatment, which demonstrated increased antimicrobial tolerance. The most active biocide against persister cells was PAA, which did not promote changes in susceptibility after their regrowth. In conclusion, persister cells are ubiquitous within biofilms and survive after critical biocide treatment. The descendant planktonic and biofilms populations showed similar properties as the original ones.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for Twelve Antimicrobials (Biocides and Antibiotics) in Eight Strains of Listeria monocytogenes

When selecting effective doses of antimicrobials, be they biocides or antibiotics, it is essential to know the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of these substances. The present research determined the MICs and MBCs for three biocides, sodium hypochlorite (SH), benzalkonium chloride (BC), and peracetic acid (PAA), and nine antibiotics in eight strains of Listeria monocytogenes of varying serotypes. Marked intra-species differences were observed in the resistance of L. monocytogenes to the biocides and antibiotics. The MICs (ppm) for the biocides ranged between 1750 and 4500 for SH, 0.25 and 20.00 for BC, and 1050 and 1700 for PAA. Their MBCs (ppm) ranged from 2250 to 4500 for SH, 0.50 to 20.00 for BC, and 1150 to 1800 for PAA. The MICs (ppm) for antibiotics lay between 1 and 15 for ampicillin, 8 and 150 for cephalothin, 20 and 170 for cefoxitin, 0.05 and 0.20 for erythromycin, 4 and 50 for chloramphenicol, 3 and 100 for gentamicin, 2 and 15 for tetracycline, 2 and 80 for vancomycin, and 160 and 430 for fosfomycin. The corresponding MBCs (ppm) were from 5 to 20 for ampicillin, 9 to 160 for cephalothin, 70 to 200 for cefoxitin, 4 to 5 for erythromycin, 9 to 70 for chloramphenicol, 5 to 100 for gentamicin, 3 to 30 for tetracycline, 3 to 90 for vancomycin, and 160 to 450 for fosfomycin. Notably, erythromycin showed considerable efficacy, demonstrated by the low values for both MIC and MBC. Based on EUCAST and the CLSI criteria, all strains were susceptible to erythromycin. All strains were resistant to cephalothin, cefoxitin, gentamicin, and fosfomycin. Further values for resistance were 87.50% for ampicillin and vancomycin, 75.00% for tetracycline, and 62.50% for chloramphenicol. The high prevalence of antibiotic resistance is a matter for concern. A positive correlation was found between MIC and MBC values for most of the biocides and antibiotics. The higher the hydrophobicity of the cell surface, the higher the susceptibility to biocides, suggesting that surface characteristics of bacterial cells influence resistance to these compounds.

The enzymatic method for the quantitative determination of benzalkonium chloride in the antiseptic solution “CUTASEPT® F”

Aim. To develop an alternative method for the quantitative determination of the benzalkonium chloride content as an active pharmaceutical ingredient in the disinfectant solution “CUTASEPT® F”.Materials and methods. The method is based on the ability of benzalkonium chloride to inhibit the enzymatic hydrolysis of acetylcholine by acetylcholinesterase. The reaction rate is assessed by the non-hydrolyzed acetylcholine residue, which is determined by the amount of peracetic acid produced during the interaction with the excess of the hydrogen peroxide solution. The indicator reaction is the interaction of p-phenetidine with peracetic acid that leads to the formation of 4,4’-azoxyphenetole with λmax = 358 nm (log10 ε = 4.2).Results and discussion. As a result of the research conducted the linear dependence of the degree of inhibition of the enzymatic hydrolysis of acetylcholine (U, %) on the concentration of benzalkonium chloride was determined in the concentration range of (0.5 – 7.0) × 10–6 mol L-1 with the correlation coefficient of 0.999. The limit of quantitation was 1.9 × 10–6 mol L-1.Conclusions. As a result of the research conducted the kinetic enzymatic method for the quantitative determination of benzalkonium chloride has been developed by its inhibitory effect in the biochemical reaction of acetylcholine hydrolysis. This method is fast, cheap and easy to perform, does not require expensive equipment, and available for use in the field.

Impact of benzalkonium chloride-preserved and preservative-free latanoprost eye drops on cultured human conjunctival goblet cells upon acute exposure and differences in physicochemical properties of the eye drops

ObjectiveTo investigate the short-term impact on human conjunctival goblet cell (GC) survival and mucin release of acute exposure to benzalkonium chloride (BAK) preserved and preservative-free (PF) 0.005% (w/v) latanoprost (LT) eye drops, and to compare the eye drops’ physicochemical properties.Methods and analysisPrimary GC cultures were established from human conjunctival donor tissue. The impact of eye drops on GC survival was assessed using a lactate dehydrogenase assay. Mucin release was evaluated through mucin-specific immunostaining. pH value, osmolality, drop mass and surface tension for all LT eye drops were measured.ResultsAfter application with PF-LT for 30 min (min), the GC survival was maintained compared with control (p=0.9941), while all BAK-LT eye drops reduced survival with approximately 30% (p<0.02). Following application with PF-LT for 30 min, mucin was found around the GC nucleus, as seen in the vehicle control, indicating no secretion. In contrast, BAK-LT caused diffuse staining of mucin, similar to the secretagogue histamine, indicating stimulation of secretion. The pH value of the BAK-LT and PF-LT eye drops were 6.0–6.9 and 6.8, respectively. The osmolality was 258–288 mOsm/kg for the BAK-LT eye drops and 276 for PF-LT eye drops. The mean drop mass was 26–31 mg for the BAK-LT eye drops and 30 mg for PF-LT. The surface tension was lower for all BAK-LT eye drops (31.1–32.1 mN/m) compared with PF-LT (42 mN/m).ConclusionPF-LT compared with various branded and generic LT preparations containing BAK are less cytotoxic when applied to cultured GCs.

Differences in Effectiveness of Antibacterial Power Between Cocoa Peel Extract (Theobroma cocoa L.) and Benzalkonium Chloride 0.1% Against Staphylococcus aureus (In Vitro)

Background: Staphylococcus aureus is a gram-positive bacteria play a role in the formation of dental biofilm which iscausing dental caries. During tooth preparation, to stop the growth of bacteria, a cavity cleaning agent is given using achemical, namely Benzalkonium Chloride (BAC) 0.1%, but BAC has disadvantages including allergic reactions, tolerantmicrobes, and resistance. Therefore, it is hoped that there will be herbal ingredients that can be used as an alternative.Cocoa peel extract has active compounds of tannins, flavonoids, alkaloids, terpenoids, and saponins which haveantibacterial concentration 6% according to safe concentrations. Purpose: To explain the difference in the effectivenessof the antibacterial power of 6% cocoa peel extract and 0.1% BAC against Staphylococcus aureus (in vitro). Methods:This study was a laboratory experimental in vitro with the posttest only control group design. Using the diffusion methodfor Staphylococcus aureus that divided into two parts, 6% cocoa peel extract and 0.1% BAC. Each petri dish was givendisc paper dripped with 0.01 ml of each test material, then incubated for two days and observed the diameter of theinhibition zone. Results: The average diameter of the inhibition zone formed in the 6% cacao peel extract was 11.5288mm and BAC 0.1% was 18.2925 mm against Staphylococcus aureus. Conclusion: There was a significant difference inthe effectiveness of antibacterial power (p <0.05) between 6% cacao peel extract (Theobroma cacao L.) and 0.1% BACagainst Staphylococcus aureus (In Vitro).

The renaissance of barrier contraception (based on the data from an international clinical trial)

Background. In recent years, interest in the use of barrier methods of contraception has increased, since many women prefer them in connection with the change in the epidemiological situation associated with COVID-19, with contraindications to other methods of contraception, as well as with irregular sex life and casual sexual intercourse. In addition, these methods of contraception are preferred for breastfeeding mothers and pre- and postmenopausal women. Studies of the efficacy of spermicidal agents in late reproductive age women have not previously been carried out, although their use in this population is of particular interest, since these agents do not have a systemic effect and, if used correctly, can prevent unwanted pregnancies. The article presents data from an international French-Russian study of the efficacy of spermicides containing benzalkonium chloride in late reproductive age women (over 40 years). The study was carried out on the basis of 7 private offices of obstetricians and gynecologists in France and 6 obstetric and gynecological clinics in Russia. Aim. Assessment of the contraceptive efficacy, safety and acceptability of a cream containing benzalkonium chloride in women over 40 years of age. Materials and methods. The study included non-pregnant women aged 40 and over who had at least 1 menstrual period in the past 3 months and who would like to use spermicides as contraception for at least 6 months. After a 6-month period of mandatory use of spermicide, patients could, if desired, continue to use it for the next 6 months. All women were given Pharmatex (1.2%), a vaginal cream containing benzalkonium chloride (1.2 g per 100 g of cream) as the active ingredient, 1 standard dose before each intercourse. The primary endpoint was the Pearl Index. The acceptability of the method after each use of the cream, the moisturizing effect, and the woman's and researcher's overall satisfaction with this method of contraception were also assessed. Safety was assessed using adverse event monitoring. Results. An analysis of efficacy of the study drug showed that during study, pregnancy did not occur in any of the women. Pearl Index for 12 months was 0. Over the mandatory 6-month period, the use of Pharmatex cream was assessed by patients as acceptable (to some extent acceptable, acceptable, completely acceptable) in 98% of sexual intercourse, and over the 12-month period in 98.6% of intercourse. The moisturizing effect of the cream was noted by 96.1% of women. In 6 months of the use of Pharmatex cream, overall satisfaction was rated as quite good, good, or very good by 99.3% of patients and in 12 months by 100% of patients. Adverse events were noted only in 0.5% of cases. Most of these events were assessed as unrelated to the study drug. Conclusion. The use of a benzalkonium chloride spermicidal cream can be considered an effective and acceptable method of contraception for women over the age of 40. It is well tolerated, has a moisturizing effect on the vaginal mucosa, and meets the needs and lifestyle of women. The contraceptive with benzalkonium chloride has a favorable safety profile: it does not adversely affect the normal flora of the vagina, can be used during breastfeeding, since it does not penetrate into the vascular bed and doesnt have systemic effects.