The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin

Abstract

Nontypeable Hemophilus influenzae (NTHi) is a Gram-negative bacterial pathogen that causes chronic biofilm infections of the ears and airways. The biofilm matrix provides structural integrity to the biofilm and protects biofilm cells from antibiotic exposure by reducing penetration of antimicrobial compounds into the biofilm. Extracellular DNA (eDNA) has been found to be a major matrix component of biofilms formed by many species of Gram-positive and Gram-negative bacteria, including NTHi. Interestingly, the cation chelator ethylenediaminetetra-acetic acid (EDTA) has been shown to reduce the matrix strength of biofilms of several bacterial species as well as to have bactericidal activity against various pathogens. EDTA exerts its antimicrobial activity by chelating divalent cations necessary for growth and membrane stability and by destabilizing the matrix thus enhancing the detachment of bacterial cells from the biofilm. In this study, we have explored the role of divalent cations in NTHi biofilm development and stability. We have utilized in vitro static and continuous flow models of biofilm development by NTHi to demonstrate that magnesium cations enhance biofilm formation by NTHi. We found that the divalent cation chelator EDTA is effective at both preventing NTHi biofilm formation and at treating established NTHi biofilms. Furthermore, we found that the matrix destablilizers EDTA and DNaseI increase the susceptibility of NTHi biofilms to ampicillin and ciprofloxacin. Our observations indicate that DNaseI and EDTA enhance the efficacy of antibiotic treatment of NTHi biofilms. These observations may lead to new strategies that will improve the treatment options available to patients with chronic NTHi infections.

In vitro and in vivo activity of EDTA and antibacterial agents against the biofilm of mucoid Pseudomonas aeruginosa

“…Indeed, Lambert et al [33] showed that EDTA had a synergistic effect with other antimicrobial agents. DNaseI and EDTA were used in combination with ampicillin and ciprofloxacin to reduce Haemophilus influenzae biofilm [34]. On the other hand, a study on otopathogenic strains of PA revealed that while EDTA delayed biofilm development and could inhibit growth of planktonic PA, it did not suppress biofilm formation in all the otopathogenic strains of PA [35].…”

Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake

“…The EM can bind various antimicrobials with its versatile components. For example, eDNA interacts with aminoglycosides, thereby protecting P. aeruginosa biofilm-associated cells [33]; sub-inhibitory concentrations of penicillin augment the production of all EM components of Actinobacillus pleuropneumoniae [34]; and the treatment of Haemophilus influenzae biofilms with DNase I makes the cells more sensitive to ampicillin and ciprofloxacin [35]. Deletion of hofQ decreased the amount of eDNA while increasing the susceptibility to the tested β-lactams, supporting the hypothesis that eDNA can sequester and protect biofilm-associated cells from the activity of β-lactams.…”

Extracellular DNA in oral microbial biofilms

“…For example, DNase I increases the susceptibility of nontypeable Haemophilus influenzae to ampicillin and ciprofloxacin and partially sensitises biofilm cells of Mycobacterium tuberculosis to isoniazid [51,52]. Vancomycin binds 100 times more tightly to DNA than to its cellular target, the dAla-d-Ala C terminus of the peptidoglycan pentapeptide, and eDNA protects Staphylococcus epidermidis biofilms from vancomycin [53].…”