Suppression of planktonic and biofilm of Escherichia coli by the synergistic lantibiotics-polymyxins combinations.

Escherichia coli are generally resistant to the lantibiotic's action (nisin and warnerin), but we have shown increased sensitivity of E. coli to lantibiotics in the presence of subinhibitory concentrations of polymyxins. Synergistic lantibiotic-polymyxin combinations were found for polymyxins B and M. The killing of cells at the planktonic and biofilm levels was observed for two collection and four clinical multidrug-resistant E. coli strains after treatment with lantibiotic-polymyxin B combinations. Thus, 24-h treatment of E. coli mature biofilms with warnerin-polymyxin B or nisin-polymyxin B leads to five to tenfold decrease in the number of viable cells, depending on the strain. AFM revealed that the warnerin and polymyxin B combination caused the loss of the structural integrity of biofilm and the destruction of cells within the biofilm. It has been shown that pretreatment of cells with polymyxin B leads to an increase of Ca2+ and Mg2+ ions in the culture medium, as detected by atomic absorption spectroscopy. The subsequent exposure to warnerin caused cell death with the loss of K+ ions and cell destruction with DNA and protein release. Thus, polymyxins display synergy with lantibiotics against planktonic and biofilm cells of E. coli, and can be used to overcome the resistance of Gram-negative bacteria to lantibiotics.

The biofilm formation of nontuberculous mycobacteria and its inhibition by essential oils.

Background: Nontuberculous mycobacteria (NTM) form two types of biofilms: Bottom biofilm and pellicle. The spatial distribution of cells between these types of biofilms and their dispersion into the liquid medium depends on the ratio of the nutrient components of the growth medium. The inhibition of biofilm formation by NTM can be achieved through the use of lipophilic compounds, such as essential oils (EOs). Method: The biofilm and pellicle formation of Mycobacterium smegmatis and Mycobacterium avium on four nutrient media under static conditions and in the vapors of six EOs was evaluated by conventional method. The antimycobacterial effect of EOs was also studied by the disc diffusion method. Results: The bottom biofilm and pellicle formation of NTM largely depended on the composition and availability of nutrients. Nutrient media in which NTM form powerful bottom biofilm or pellicle or both have been determined. The growth of studied NTM strains on agar was highly sensitive to the EOs of Scots pine, Atlas cedar, bergamot, and a mixture of EO of different plants. The cultivation of bacteria in the EO vapors also resulted in total suppression of the pellicle for all studied NTM strains. Conclusions: Our data clearly indicate that the carbon-nitrogen ratio is involved in the regulation of the spatial distribution of the biofilm. The preventing effect of EOs vapors, especially the synergistic action of mixture of EOs on the biofilm and pellicle formation by NTMs can be observed.

VapBC and MazEF toxin/antitoxin systems in the regulation of biofilm formation and antibiotic tolerance in nontuberculous mycobacteria.

Background: Mycobacterium smegmatis and other nontuberculous mycobacteria (NTM) are widely distributed in the environment, but a significant increase of NTM infections has taken place in the last few decades. The objective of this study was to determine the role of toxin-antitoxin (TA) vapBC and mazEF systems that act as effectors of persistence in the stress response of NTM. Methods: The growth ability and the biofilm formation of NTM were evaluated by conventional methods. Bacterial cell viability was determined using MTT staining, agar plating, or the method of limiting dilutions. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antibiotics were estimated using broth and agar dilution methods. Results: Despite a comparable growth dynamics and biofilm formation on solid/liquid interface with the wild type, a M. smegmatis vapBC, mazEF, and vapBC × mazEF deletion mutant produced more abundant pellicle and were more susceptible to heat shock. Significant differences were also found in the resistance wild type of NTM to isoniazid and ciprofloxacin reflected by higher MBC/MIC ratios. The proposed method of cultivation of agar blocks without visible growth after MIC determination into a liquid medium allows us to detect transition of all wild type of NTM strains to a dormant state in the presence of subMICs of isoniazid and ciprofloxacin while all deletion mutants failed to form dormant cells. Conclusion: Our data suggest that both vapBC and mazEF TA systems putatively involved in the heat and antibiotic stress response of NTM via their key role in transition to the dormant state.

The effect of sucrose-induced osmotic stress on the sensitivity of Escherichia coli to bacteriocins.

Bacteriocins are antimicrobial peptides, produced by Gram-positive bacteria such as lactococci and staphylococci, that have limited bactericidal action against Gram-negative bacteria. The aim of this paper was to study the sensitivity of three strains of Escherichia coli to bacteriocins: nisin (as Nisaplin®) and two staphylococcal peptides (warnerin and hominin) during sucrose-induced osmotic stress. We found that all peptides in a 0.3 g·mL-1 sucrose solution significantly reduced the number of viable E. coli. The most pronounced antibacterial effect was achieved by nisin against E. coli K-12 (3 log reduction). Slightly less bactericidal effects were observed with warnerin (1 mg·mL-1) and hominin (1 mg·mL-1) in sucrose solution. The lytic activity of staphylococcal peptides was detected by decreased optical density and viable cell counts. Moreover, it was confirmed by the increased amount of DNA and protein in the medium and the morphological changes detected by atomic force microscopy after 20 h of treatment. Zymographic analysis revealed the release of lytic enzymes from E. coli cells after treatment with staphylococcal peptides and sucrose. These results indicated that the antimicrobial action of peptides can be extended to Gram-negative bacteria via combination with high concentrations of sucrose.

Plasma, serum, albumin, and divalent metal ions inhibit the adhesion and the biofilm formation of Cutibacterium (Propionibacterium) acnes.

Adhesion and biofilm formation of human skin bacteria C. acnes on plasma, serum and albumin-coated polystyrene or in the presence of these blood components were studied. The proteins which were pre-adsorbed to polystyrene surface or added to the medium simultaneously with bacterial cells reduced C. acnes adhesion and biofilm formation by 2-5 times to compare to the control. The role of calcium, magnesium and zinc on C. acnes attachment was also assessed. Calcium (1 and 10 mM) had the inhibitory effect on C. acnes adhesion, whereas zinc (1 and 10 mM) diminished the biofilm formation of C. acnes. We also observed that C. acnes cells did not bind to erythrocytes. Thus, we suggest that bacteria C. acnes preferably colonize the plasma-poor environment due to the inhibitory effect of blood components, in particular, albumin, calcium, and zinc.

N-acetylcysteine inhibits growth, adhesion and biofilm formation of Gram-positive skin pathogens.

N-Acetylcysteine (NAC) is a non-antibiotic drug with antimicrobial properties against biofilm phenotypes of Gram-positive and Gram-negative bacteria. Our aim was to assess the effects of NAC on the growth of Gram-positive human skin and mucous membrane pathogens in the planktonic and biofilm phases. The minimum inhibitory concentrations (MICs) of NAC against Enterococcus faecalis, Corynebacterium ammoniagenes, Mycobacterium smegmatis, Propionibacterium acnes, Staphylococcus aureus, S. epidermidis, Streptococcus pyogenes, and 14 clinical strains of coagulase-negative staphylococci (CNS) ranged from 0.098 to 25 mg/ml. We found that at sub-MICs of NAC the adherence of E. faecalis, S. epidermidis, and nine CNS strains significantly reduced. However, biofilm formation of E. faecalis, S. aureus and two CNS strains increased at sub-MICs of NAC. Furthermore, a dose-related decrease in biofilm formation of C. ammoniagenes, M. smegmatis, P. acnes, S. pyogenes, and S. epidermidis was observed. The effect of NAC on planktonic growth and biofilm formation of the M. smegmatis cell was also time-dependent. We have selected P. acnes VKM Ac-1450 Rifr strain with total resistance to rifampicin and used this microorganism for multispecies P. acnes - S. epidermidis biofilm model. The biofilm formation and growth of mixed culture of P. acnes and S. epidermidis was significantly slowed at 12.5 mg/ml of NAC. NAC also has a higher disruptive effect on both mature M. smegmatis and mixed P. acnes - S. epidermidis biofilm. Thus, NAC appears to be a promising, non-antibiotic alternative to prevent biofilm-associated infections.