This study was aimed at verifying the potential of some α-helical AMPs as lead compounds for the development of novel antimicrobials to treat lung disease in CF patients. To this, we tested the in vitro susceptibility of P. aeruginosa, S. maltophilia and S. aureus CF isolates to the naturally occurring AMPs BMAP-27 and BMAP-28, as well as the rationally designed P19(B/9), and we compared their effectiveness with that of Tobramycin, the antibiotic of choice for the inhalation therapy of chronic airway infections in CF patients.
BMAP-27 and BMAP-28 are two cathelicidin-derived peptides of bovine origin that have a role in innate defence [27
]. The hallmark of cathelicidins is the presence of a conserved N-terminal proregion associated with C-terminal antimicrobial sequences showing a remarkable diversity and considerable inter-species differences [13
]. BMAP-27 and BMAP-28 are cationic (charge: +11 and +8, respectively) and both adopt an α-helical structure on interaction with the negatively charged bacterial surface [28
]. Recent results have suggested that AMPs with these characteristics may be the most effective against strains producing exogenous polysaccharides that are known to inhibit the activity of other types of AMPs [19
]. For this reason, we added to our study also a third peptide from this class which has been rationally designed, making use also of non-proteinogenic aminoacids, to optimize its propensity to assume α-helical conformation [30
Effort to treat CF are also hampered by the conditions present in patients’ airway surface liquid where the accumulation of large volumes of viscous sputum (mucus) providing bacteria with a nutritionally rich growth environment composed of host- and bacterial-derived factors which deeply change their phenotype and possibly their susceptibility against AMPs [31
]. Therefore, to accurately judge the feasibility of these peptides as potential anti-infectives in the context of CF, in this study we investigated the activity of AMPs under some CF-like experimental conditions, including acidic pH, reduced O2
tension, and a chemically defined medium mimicking the nutritional composition of CF sputum [24
These conditions allow pathogens to assume a physiology similar to that shown in vivo
in the CF lung [24
] and constitute a more realistic model to assay their sensitivity to AMPs.
Evaluation of MIC and MBC values, as well as time-killing assays against planktonic forms of different CF isolates of P. aeruginosa, S. maltophilia, and S. aureus, have shown that all three AMPs are highly active in vitro against most tested strains, although BMAP-28 showed the widest spectrum of activity. It is noteworthy that all the three peptides exhibited an activity higher than Tobramycin. This observation is even more evident when considering the molar concentration (μM) of each compound rather than that by weight (μg/ml), given that the peptides tested are at least six folds heavier than Tobramycin.
The poor activity showed by Tobramycin is probably due to the experimental conditions used in this study, as suggested by comparative evaluation of MIC values observed in both “CF-like” and CLSI-recommended conditions. On the contrary, the activity of AMPs tested resulted to be slightly enhanced (BMAP-28), unaffected (BMAP-27), or slightly reduced [P19(9/B)] in “CF-like” conditions, compared to CLSI-recommended ones, so they can be considered to be quite robust and medium insensitive.
MBC/MIC ratio clearly indicated that all AMPs exert a bactericidal effect against the CF isolates, in agreement with the known capability of BMAP-27, BMAP-28 and P19(B/9) to kill target cells by rapid permeabilization of their membranes [28
]. Results of killing kinetic assays confirmed this mode of action, although bactericidal activity against S. aureus
and S. maltophilia
was strain-dependent. Again, the potency of AMPs was overall comparable or higher than that showed by Tobramycin.
Due to the different mechanism of action showed by AMPs and Tobramycin, we investigated the potential synergy between them. Interestingly, Tobramycin exhibited synergy with both BMAP-27 and P19(9/B) against planktonic S. aureus Sa4 and Sa10 strains, both resistant to Tobramycin, thus suggesting that at least in these cases both AMPs may overcome resistance to Tobramycin by facilitating the internalization of the aminoglycoside into the bacterial cells. Further studies on a more representative number of S. aureus strains will be mandatory to understand the mechanism of this synergy and the feasibility to use these AMPs in association with traditional antibiotic treatments.
Within the CF lung, pathogens cells grow as biofilms, which are inherently recalcitrant to antimicrobial treatment and host response [32
]. Even worse, it has recently been reported that some antibiotics may even stimulate biofilm formation at subinhibitory concentrations [7
]. Biofilm resistance is mainly due to the slow growth rate and low metabolic activity of bacteria in such community. For these reasons, AMPs whose mechanism of action makes them active also on non-growing bacteria, should be able to efficiently inhibit or prevent biofilm formation.
Our results in fact indicate that the three α-helical peptides were all able to reduce biofilm formation, although generally at a less extent than Tobramycin. In particular, all peptides reduced the capacity of P. aeruginosa, S. maltophilia
and S. aureus
to form biofilms when used at sub-inhibitory concentrations, with the strongest effects at about 1/2xMIC values, while Tobramycin was efficacious also at lower concentrations (1/4x, and 1/8x MIC). This effect was particular evident with the isolates of S. aureus
. Interestingly, no planktonic growth inhibition was observed at concentrations able to reduce biofilm formation, and also AMPs with poor killing capacity against some planktonic cells showed anti-biofilm effects. These observations suggest that BMAP-27, BMAP-28 and P19(9/B) may interfere with biofilm formation by different mechanisms other than direct antimicrobial activity similarly to what observed with the human cathelicidin LL-37 [33
], and recently reviewed by Batoni et al. [34
Most CF patients are infected by P. aeruginosa
whose persistence is due to the formation of antibiotic resistant biofilms in the lung [35
]. Our results showed that BMAP-27, BMAP-28, and P19(9/B) were also as effective as Tobramycin in reducing cell viability of preformed biofilms formed by selected strains of P. aeruginosa.
At MIC concentrations, and even more at 5xMIC values, the two cathelicidins caused highly significant reduction of biofilm viability of all six strains of P. aeruginosa
whereas Tobramycin showed comparable results only for five isolates. It has previously been reported that extracellular DNA is an important biofilm component [36
], and that in P. aeruginosa
it is involved in cell-cell attachment and biofilm development [37
]. Due to the high affinity of cationic AMPs for DNA [38
], it may be presumed that this binding might facilitate the detachment or disruption of otherwise-stable biofilm structures.