Lactoferrin disaggregates pneumococcal biofilms and inhibits acquisition of resistance through its DNase activity

Streptococcus pneumoniae colonizes the upper airways of children and the elderly. Colonization progresses to persistent carriage when S. pneumoniae forms biofilms, a feature required for the development of pneumococcal disease. Nasopharyngeal biofilms are structured with a matrix that includes extracellular DNA (eDNA), which is sourced from the same pneumococci and other bacteria. This eDNA also allows pneumococci to acquire new traits, including antibiotic resistance genes. In this study, we investigated the efficacy of lactoferrin (LF), at physiological concentrations found in secretions with bactericidal activity [i.e., colostrum (100 μM), tears (25 μM)], in eradicating pneumococcal biofilms from human respiratory cells. The efficacy of synthetic LF-derived peptides was also assessed. We first demonstrated that LF inhibited colonization of S. pneumoniae on human respiratory cells without affecting the viability of planktonic bacteria. LF-derived peptides were, however, bactericidal for planktonic pneumococci but they did not affect viability of pre-formed biofilms. In contrast, LF (40 and 80 μM) eradicated pneumococcal biofilms that had been pre-formed on abiotic surfaces (i.e., polystyrene) and on human pharyngeal cells, as investigated by viable counts and confocal microscopy. LF also eradicated biofilms formed by S. pneumoniae strains with resistance to multiple antibiotics. We investigated whether treatment with LF would affect the biofilm structure by analyzing eDNA. Surprisingly, in pneumococcal biofilms treated with LF, the eDNA was absent in comparison to the untreated control (∼10 μg/ml) or those treated with LF-derived peptides. EMSA assays showed that LF binds S. pneumoniae DNA and a time-course study of DNA decay demonstrated that the DNA is degraded when bound by LF. This LF-associated DNase activity inhibited acquisition of antibiotic resistance genes in both in vitro transformation assays and in a life-like bioreactor system. In conclusion, we demonstrated that LF eradicates pneumococcal-colonizing biofilms at a concentration safe for humans and identified a LF-associated DNAse activity that inhibited the acquisition of resistance

Bactericidal effect of bovine lactoferrin and synthetic peptide lactoferrin chimera in Streptococcus pneumoniae and the decrease in luxS gene expression by lactoferrin

Streptococcus pneumoniae (pneumococcus) is responsible for nearly one million child deaths annually. Pneumococcus causes infections such as pneumonia, otitis media, meningitis, and sepsis. The human immune system includes antibacterial peptides and proteins such as lactoferrin (LF), but its activity against pneumococcus is not fully understood. The aim of this work was to evaluate the bactericidal effect of bovine lactoferrin (bLF) and the synthetic LF-peptides lactoferricin (LFcin17-30), lactoferrampin (LFampin265-284), and LFchimera against S. pneumoniae planktonic cells. The mechanism of damage was also investigated, as well as the impact of these peptides on the transcription levels of genes known to encode important virulence factors. S. pneumoniae planktonic cells were treated with bLF, LFcin17-30, LFampin265-284 and LFchimera at different time points. The viability of treated planktonic cells was assessed by dilution and plating (in CFU/ml). The interaction between LF and LF-peptides coupled to fluorescein was visualized using a confocal microscope and flow cytometry, whereas the damage at structural levels was observed by electron microscopy. Damage to bacterial membranes was further evaluated by membrane permeabilization by use of propidium iodide and flow cytometry, and finally, the expression of pneumococcal genes was evaluated by qRT-PCR. bLF and LFchimera were the best bactericidal agents. bLF and peptides interacted with bacteria causing changes in the shape and size of the cell and membrane permeabilization. Moreover, the luxS gene was down-regulated in bacteria treated with LF. In conclusion, LF and LFchimera have a bactericidal effect, and LF down-regulates genes involved in the pathogenicity of pneumococcus, thus demonstrating potential as new agents for the treatment of pneumococcal infections