Dual-function antimicrobial-antibiofilm peptide hybrid to tackle biofilm-forming Staphylococcus epidermidis.

BACKGROUND: Due to their resistance and difficulty in treatment, biofilm-associated infections are problematic among hospitalized patients globally and account for 60% of all bacterial infections in humans. Antibiofilm peptides have recently emerged as an alternative treatment since they can be effectively designed and exert a different mode of biofilm inhibition and eradication. METHODS: A novel antibiofilm peptide, BiF, was designed from the conserved sequence of 18 α-helical antibiofilm peptides by template-assisted technique and its activity was improved by hybridization with a lipid binding motif (KILRR). Novel antibiofilm peptide derivatives were modified by substituting hydrophobic amino acids at positions 5 or 7, and both, with positively charged lysines (L5K, L7K). These peptide derivatives were tested for antibiofilm and antimicrobial activities against biofilm-forming Staphylococcus epidermidis and multiple other microbes using crystal violet and broth microdilution assays, respectively. To assess their impact on mammalian cells, the toxicity of peptides was determined through hemolysis and cytotoxicity assays. The stability of candidate peptide, BiF2_5K7K, was assessed in human serum and its secondary structure in bacterial membrane-like environments was analyzed using circular dichroism. The action of BiF2_5K7K on planktonic S. epidermidis and its effect on biofilm cell viability were assessed via viable counting assays. Its biofilm inhibition mechanism was investigated through confocal laser scanning microscopy and transcription analysis. Additionally, its ability to eradicate mature biofilms was examined using colony counting. Finally, a preliminary evaluation involved coating a catheter with BiF2_5K7K to assess its preventive efficacy against S. epidermidis biofilm formation on the catheter and its surrounding area. RESULTS: BiF2_5K7K, the modified antibiofilm peptide, exhibited dose-dependent antibiofilm activity against S. epidermidis. It inhibited biofilm formation at subinhibitory concentrations by altering S. epidermidis extracellular polysaccharide production and quorum-sensing gene expression. Additionally, it exhibited broad-spectrum antimicrobial activity and no significant hemolysis or toxicity against mammalian cell lines was observed. Its activity is retained when exposed to human serum. In bacterial membrane-like environments, this peptide formed an α-helix amphipathic structure. Within 4 h, a reduction in the number of S. epidermidis colonies was observed, demonstrating the fast action of this peptide. As a preliminary test, a BiF2_5K7K-coated catheter was able to prevent the development of S. epidermidis biofilm both on the catheter surface and in its surrounding area. CONCLUSIONS: Due to the safety and effectiveness of BiF2_5K7K, we suggest that this peptide be further developed to combat biofilm infections, particularly those of biofilm-forming S. epidermidis.

Cymbopogon citratus L. essential oil as a potential anti-biofilm agent active against antibiotic-resistant bacteria isolated from chronic rhinosinusitis patients.

Chronic rhinosinusitis (CRS) is long-term inflammation of the sinuses that can be caused by infection due to antibiotic-resistant bacteria. Biofilm developed by microbes is postulated to cause antibiotic treatment failure. Thus, the anti-biofilm activities of seven Thai herbal essential oils (EOs) against antibiotic-resistant bacteria isolated from CRS patients was investigated. Lemongrass (Cymbopogon citratus L.) EO showed the most effective antibiofilm activity against Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus epidermidis grown as biofilm. GC-MS analysis found that myrcene was the major bioactive compound. Pretreatment with lemongrass EO significantly inhibited biofilm formation of all bacterial strains in more than 50% of cases. Furthermore, confocal microscopy analysis revealed the biofilm-disrupting activity of lemongrass EO against the biofilm matrix of all these bacterial species and also increased P. aeruginosa swarming motility with no toxicity to human cells. These results suggest that lemongrass EO has promising clinical applications as an anti-biofilm agent for CRS patients.

Vitamin D (1α,25(OH)2D3) supplementation minimized multinucleated giant cells formation and inflammatory response during Burkholderia pseudomallei infection in human lung epithelial cells.

Melioidosis is an infectious disease with high mortality rates in human, caused by the bacterium Burkholderia pseudomallei. As an intracellular pathogen, B. pseudomallei can escape from the phagosome and induce multinucleated giant cells (MNGCs) formation resulting in antibiotic resistance and immune evasion. A novel strategy to modulate host response against B. pseudomallei pathogenesis is required. In this study, an active metabolite of vitamin D3 (1α,25-dihydroxyvitamin D3 or 1α,25(OH)2D3) was selected to interrupt pathogenesis of B. pseudomallei in a human lung epithelium cell line, A549. The results demonstrated that pretreatment with 10-6 M 1α,25(OH)2D3 could reduce B. pseudomallei internalization to A549 cells at 4 h post infection (P < 0.05). Interestingly, the presence of 1α,25(OH)2D3 gradually reduced MNGC formation at 8, 10 and 12 h compared to that of the untreated cells (P < 0.05). Furthermore, pretreatment with 10-6 M 1α,25(OH)2D3 considerably increased hCAP-18/LL-37 mRNA expression (P < 0.001). Additionally, pro-inflammatory cytokines, including MIF, PAI-1, IL-18, CXCL1, CXCL12 and IL-8, were statistically decreased (P < 0.05) in 10-6 M 1α,25(OH)2D3-pretreated A549 cells by 12 h post-infection. Taken together, this study indicates that pretreatment with 10-6 M 1α,25(OH)2D3 has the potential to reduce the internalization of B. pseudomallei into host cells, decrease MNGC formation and modulate host response during B. pseudomallei infection by minimizing the excessive inflammatory response. Therefore, 1α,25(OH)2D3 supplement may provide an effective supportive treatment for melioidosis patients to combat B. pseudomallei infection and reduce inflammation in these patients.

Induction of apoptosis in human colorectal cancer cells by nanovesicles from fingerroot (Boesenbergia rotunda (L.) Mansf.).

Colorectal cancer is the leading cause of cancer-related deaths worldwide, warranting the urgent need for a new treatment option. Plant-derived nanovesicles containing bioactive compounds represent new therapeutic avenues due to their unique characteristics as natural nanocarriers for bioactive molecules with therapeutic effects. Recent evidence has revealed potential anticancer activity of bioactive compounds from Boesenbergia rotunda (L.) Mansf. (fingerroot). However, the effect and the underlying mechanisms of fingerroot-derived nanovesicles (FDNVs) against colorectal cancer are still unknown. We isolated the nanovesicles from fingerroot and demonstrated their anticancer activity against two colorectal cancer cell lines, HT-29 and HCT116. The IC50 values were 63.9 ± 2.4, 57.8 ± 4.1, 47.8 ± 7.6 µg/ml for HT-29 cells and 57.7 ± 6.6, 47.2 ± 5.2, 34 ± 2.9 µg/ml for HCT116 cells at 24, 48, and 72 h, respectively. Interestingly, FDNVs were not toxic to a normal colon epithelial cell line, CCD 841 CoN. FDNVs exhibited selective uptake by the colorectal cancer cell lines but not the normal colon epithelial cell line. Moreover, dose- and time-dependent FDNV-induced apoptosis was only observed in the colorectal cancer cell lines. In addition, reactive oxygen species levels were substantially increased in colorectal cancer cells, but total glutathione decreased after treatment with FDNVs. Our results show that FDNVs exhibited selective anticancer activity in colorectal cancer cell lines via the disruption of intracellular redox homeostasis and induction of apoptosis, suggesting the utility of FDNVs as a novel intervention for colorectal cancer patients.

D-LL-31 enhances biofilm-eradicating effect of currently used antibiotics for chronic rhinosinusitis and its immunomodulatory activity on human lung epithelial cells.

Chronic rhinosinusitis (CRS) is a chronic disease that involves long-term inflammation of the nasal cavity and paranasal sinuses. Bacterial biofilms present on the sinus mucosa of certain patients reportedly exhibit resistance against traditional antibiotics, as evidenced by relapse, resulting in severe disease. The aim of this study was to determine the killing activity of human cathelicidin antimicrobial peptides (LL-37, LL-31) and their D-enantiomers (D-LL-37, D-LL-31), alone and in combination with conventional antibiotics (amoxicillin; AMX and tobramycin; TOB), against bacteria grown as biofilm, and to investigate the biological activities of the peptides on human lung epithelial cells. D-LL-31 was the most effective peptide against bacteria under biofilm-stimulating conditions based on IC50 values. The synergistic effect of D-LL-31 with AMX and TOB decreased the IC50 values of antibiotics by 16-fold and could eliminate the biofilm matrix in all tested bacterial strains. D-LL-31 did not cause cytotoxic effects in A549 cells at 25 µM after 24 h of incubation. Moreover, a cytokine array indicated that there was no significant induction of the cytokines involving in immunopathogenesis of CRS in the presence of D-LL-31. However, a tissue-remodeling-associated protein was observed that may prevent the progression of nasal polyposis in CRS patients. Therefore, a combination of D-LL-31 with AMX or TOB may improve the efficacy of currently used antibiotics to kill biofilm-embedded bacteria and eliminate the biofilm matrix. This combination might be clinically applicable for treatment of patients with biofilm-associated CRS.

DNase-mediated eDNA removal enhances D-LL-31 activity against biofilms of bacteria isolated from chronic rhinosinusitis patients.

Chronic rhinosinusitis (CRS) is a chronic infection of the nasal cavity and paranasal sinuses associated with the presence of a microbial biofilm. Extracellular DNA (eDNA) is an important component of the biofilm matrix. Antimicrobial peptides (AMPs) are natural peptides with the ability to kill microorganisms. D-LL-31 is a synthetic variant of the AMP cathelicidin with increased resistance to proteolytic breakdown. In this study it is shown for 3 clinical CRS isolates that treatment of 24 h biofilms with DNase I enhanced the antimicrobial activity of D-LL-31. Conversely, co-incubation of D-LL-31 at the IC50 value with exogenous DNA resulted in reduced antimicrobial activity. DNase I alone did not show antimicrobial activity against the isolates tested but caused dispersal of an established biofilm. Hence, the presence of eDNA in the biofilm matrix reduced AMP-mediated killing. These results suggest that combination therapy with proteolysis resistant AMP D-LL-31 and DNase could be considered for effective treatment of CRS.

D-LL-31 in combination with ceftazidime synergistically enhances bactericidal activity and biofilm destruction in Burkholderia pseudomallei.

Melioidosis is a severe disease caused by Burkholderia pseudomallei. The biofilm of B. pseudomallei acquires resistance to several antibiotics and may be related to relapse in melioidosis patients. Here, the killing activity of antimicrobial peptides (LL-37, LL-31) and the D-enantiomers (D-LL-37, D-LL-31) in combination with ceftazidime (CAZ) against B. pseudomallei 1026b, H777 and a biofilm mutant M10, derived from H777 grown under biofilm-stimulating conditions was observed. Using static conditions, D-LL-31 exhibited the strongest killing activity against the three isolates in a dose-dependent manner. IC50 values for D-LL-31 ranged from 1 to 6 µM, for isolates M10, H777, and 1026b, respectively. Moreover, D-LL-31 combined with CAZ synergistically decreased the IC50 values of the peptide and antibiotic and caused also disruption of biofilms of B. pseudomallei 1026b under flow conditions. Thus a combination of D-LL-31 and CAZ may enhance the efficacy of the currently used antibiotic treatments against B. pseudomallei.