Pseudomonas aeruginosa is a relevant pathogen causing human and animal infections. In humans, severe P. aeruginosa infections usually occur in immunocompromised patients and in nosocomial setting. P. aeruginosa infection often follow surgery or invasive procedures and causes mainly pneumonia and septicaemia. P. aeruginosa may also cause mild illnesses in healthy people, in which skin, ear and eye infections can occur. Moreover, P. aeruginosa is the major pathogen in the cystic fibrosis (CF). In CF, chronic P. aeruginosa infections occur in up to 85% of CF patients and the P. aeruginosa strains involved develop antibiotic resistance and phenotypic changes, from first infection to chronic infection and mucoid phenotype. These phenotypical changes could play a major role in the persistence of P. aeruginosa infections in CF patients [1]. Antibiotic resistance and the persistence of the organisms despite therapy once chronic infection has been established, is leading to the search for more effective therapeutic approaches [1].

Pseudomonas aeruginosa also cause diseases in both livestock and companion animals, including otitis and urinary tract infections in dogs, mastitis in dairy cows and endometritis in horses [2]. Resistance phenotypes are more frequent in dogs and multi-drug resistant (MDR) P. aeruginosa seem to emerge mainly in those suffering from otitis. Antimicrobial resistance in animal P. aeruginosa infections should be closely monitored in the future, in line with possible animal-to-human transfers between pets and owners [2]. P. aeruginosa is naturally resistant to many classes of drugs and its capacity to rapidly develop resistance during treatment is a frequent source of therapeutic failures. P. aeruginosa is one of the six ESKAPE pathogens, reported by the Infectious Diseases Society of America, that urgently require novel therapies [3]. Rates of antibiotic resistance in P. aeruginosa are increasing worldwide even if the true frequency of infections caused by MDR P. aeruginosa is difficult to estimate. A review of studies reporting on MDR, extensively-drug resistant (XDR) and pan-drug resistant (PDR) P. aeruginosa infections revealed that aminoglycosides, antipseudomonal penicillins, cephalosporins, carbapenems and fluoroquinolones [4] have become ineffective as first line agents. The multidrug resistance of P. aeruginosa could be mediated by several mechanisms including multidrug efflux systems, enzyme production, outer membrane protein loss and target mutations [5]. The spread of antimicrobial resistance increase human and animal health hazard worldwide, thus makes mandatory the investigation of novel approaches to cover the therapeutic shortfall. In this view, one of the actions put forward in the European Commission Action Plan is to develop effective antimicrobials or alternatives for treatment of human and animal infections and to reinforce research to develop innovative means to combat antimicrobial resistance [6]. Antimicrobial peptides offer potential advantages over currently used classes of drugs. They may counteract pathogenic challenge by rapid, broad spectrum, microbicidal activity [7], targeting multiple pathogens with one treatment. Moreover, antimicrobial peptides may have the potential to ultimately reduce the rate of emergence of resistant microorganisms, since selective pressure is not focused to a single specific molecular target. Further, antimicrobial peptides could also be potentially used in conjunction with conventional antibiotics as part of a “combination therapy” to create an additive or synergistic effect.

The antimicrobial peptide AMP2041 is a cyclic antimicrobial peptide, belonging to a novel family of antimicrobial cationic peptides, which showed good antimicrobial activity against a panel of different Gram-positive and Gram-negative bacterial pathogens of animal origin [8]. The activity of AMP2041 against P. aeruginosa ATCC 27853 was also demonstrated, as well as additivity in combination with levofloxacin [9]. We hypothesized that the reported antimicrobial activity derived from a bacterial membrane damage, but a direct membrane damage was not previously investigated for P. aeruginosa.

The aim of the present work was to evaluate the antimicrobial activity of AMP2041 on different MDR, PDR and XDR P. aeruginosa clinical isolates of human origin, including five different phenotypes of P. aeruginosa derived from a single patient with cystic fibrosis, and on clinical MDR P. aeruginosa clinical isolates deriving from animals, mainly dogs with otitis. Further, we investigated the evidence for a direct membrane damage on P. aeruginosa ATCC 27853 reference strain.

Pseudomonas aeruginosa is an ubiquitous organism. Its ability to survive on minimal nutritional requirements and to tolerate a variety of physical conditions allows its persistence in both community and hospital settings [12]. P. aeruginosa is a serious therapeutic challenge for treatment of both community-acquired and nosocomial infections, due to the ability of this microorganism to develop resistance to multiple classes of antibacterial agents, even during the course of therapy [13, 14]. The increasing frequency of MDR or XDR P. aeruginosa strains is of concern as effective antimicrobial options are limited [15, 16]. Moreover, only a few new antibiotics are currently under development [6]. An increase in MDR bacterial infections among companion animals has been documented in multiple veterinary hospital settings [17]. This is of particular importance due to the risk of transmission to humans and other companion animals in close contact with infected animals, even because in our countries the pet population continues to rise and the contacts between people and their companion animals grows stronger [18–20]. Therefore, the discover of new agents or innovative approaches able to counteract the growing problem of antimicrobial resistance become crucial.

The synthetic peptide AMP2041 is a cationic peptide and possesses a significant proportion of hydrophobic or non-polar residues. These structural features are common to many antimicrobial peptides [9, 21]. The hydrophobic core is essential for the antimicrobial peptide to effectively permeate the bacterial membrane. The hydrophobic core is flanked at both ends by cationic and polar residues that help to solubilize the peptides in aqueous solution. Cationic and polar residues are also important for the initial electrostatic attraction of antimicrobial peptides to negatively charged phospholipid membranes of bacteria. Also the conformation assumed by AMP2041 might be responsible for the observed antimicrobial activity [8, 9, 22].

Antimicrobial activity of AMP2041 on human clinical isolates was never investigated before. In this study we evaluated the activity of AMP2041 on 19 MDR or XDR P. aeruginosa strains isolated from different pathological conditions of humans, among which five deriving from a CF patient. Moreover, the activity of AMP2041 was tested on a sample of 30 MDR P. aeruginosa strains derived from dog otitis. AMP2041 showed an excellent activity against all the examined strains. In particular, on average, it was more effective against animals strains, with an LD₉₀ of 1.69 μg/ml and an MBC of 6.4 μg/ml (3.2 μM), compared with human strains, LD₉₀ of 3.3 μg/ml, MBC of 12.5 μg/ml (6.2 μM). The antimicrobial activity found here for AMP2041 against P. aeruginosa is comparable or better than many highly-active antimicrobial peptides. Zhou et al. have found MIC values against P. aeruginosa ranging from 31 to >256 μg/ml for peptides synthesized via ring-opening polymerization of α-amino acid N-carboxyanhydrides [23]. Regarding the activity of Cecropin A, an insect antimicrobial peptide, against P. aeruginosa, a MIC value of 64 μg/ml is reported by Zhou et al. [23] and a lethal concentration of 3.5 μM by Andreu et al. [24]. For PR-39, an antimicrobial peptide from pig intestine, a lethal concentration of 200 μM against P. aeruginosa was reported [25]. Very recently, minimum lethal concentrations ranging from 3 to 100 μM were reported for E. coli MreB derived antimicrobial peptides against P. aeruginosa [26].

It is noteworthy the antimicrobial activity of AMP2041 against the strains derived from the patient with CF. Most patients with CF become chronically infected with wild-type (first infection) P. aeruginosa strains early in their life. During the years following the initial colonization, the first infection strains may mutate into mucoid variants [27, 28]. Conversion to the mucoid phenotype is thought to be driven mainly by the unique CF microenvironment [28]. In our case, the P. aeruginosa mucoid strain was less sensitive to AMP2041 than the other tested CF strains (Fig. 1b). However, this result was obtained on a single strain and should be further investigated with a wider sample to confirm a higher resistance of the mucoid phenotype compared to the first and chronic infection isolates. The observed lower sensitivity to AMP2041 of the P. aeruginosa mucoid strain could be linked to the over production of mucoid exopolysaccharides that hide the negatively charged surface components to which positively charged peptides are attracted.

Mean MBC for dog strains (n = 30, MBC = 6.4 μg/ml—see Fig. 1a) is higher than values previously found for the reference strain ATCC 27853 (4.35 μg/ml) and other dog isolates (n = 6, MBC = 2.44 μg/ml) [8]. Therefore, the increased sample size allowed us to re-evaluate the MBC average value previously obtained.

The bacterial killing assay indicated a CFU reduction >90% within 20 min (Fig. 1c). Therefore, the antimicrobial activity of AMP2041 occurs quickly and the killing kinetic profiles of human and animal clinical isolates, never investigated before, were similar to that previously reported for P. aeruginosa ATCC 27853 [8, 9]. These results were almost unrelated to the sources of strains, suggesting that the mechanism of action was similar for all the examined strains. The killing kinetics are comparable [29] or better [30] than those obtained with other established antimicrobial peptides at their lethal concentration. Saikia et al., instead, showed that two out of four E. coli MreB derived peptides completely killed P. aeruginosa within 5 min of treatment with the peptides at their minimum lethal concentrations [26]. However, in our case the kinetic of killing of AMP2041 was derived from the testing of many different P. aeruginosa strains, while in the other cases only one P. aeruginosa strain was tested. Moreover, an interesting fact that emerges from the work of Saikia et al. is that for P. aeruginosa there is no direct correlation between the minimum lethal concentration and the rapidity of killing, because the peptide with the best minimum lethal concentration (3 μM) completely killed the bacteria only after 120 min.

The stain-dead assay was performed on the P. aeruginosa ATCC 27853 reference strain and on PA-H 24 and PA-VET 38, which were selected for the assay as representative of strains with a high level of antibiotic resistance, being XDR and MDR (see Tables 1, 3), respectively. Results indicated that the inhibitory effect of AMP2041 is linked to an altered permeability of the cellular membrane of P. aeruginosa (Fig. 2). This is in accordance with the mechanism of action of cationic antimicrobial peptides which cause cell death through loss of membrane integrity [22]. The timing of the occurrence of red fluorescence was in accordance with time kill results. The membrane damage was evident within 10 min of incubation for the reference strain and the animal isolate PA-VET 38 and within 15 min for the human isolate PA-H 24.

To confirm that the fluorescence increase was due to morphological changes of bacterial membrane, a SEM analysis was performed on the P. aeruginosa ATCC 27853 reference strain treated with AMP2041. SEM analysis provided evidence for a direct membrane damage, showing the presence of several holes, dents and bursts throughout cell wall (Fig. 3b, c). Similar membrane changes are also described for other cationic antimicrobial peptides [23, 26, 31]. The microbicidal effect of AMP2041 was also confirmed by the presence of lysed cells.

In conclusion, we assessed the good antimicrobial activity of AMP2041 on P. aeruginosa strains of human origin, including those deriving from a CF patient. Moreover, we confirmed the excellent antimicrobial activity of AMP2041 on P. aeruginosa strains derived from dog otitis. We also assessed that AMP2041 antimicrobial activity is linked to changes of the P. aeruginosa cell wall morphology and the increasing of membrane permeability. This mechanism of action is less prone to induce resistance by the pathogen compared to antimicrobials acting against intracellular targets. However, clinical trials with adequate animal models should be performed to define the therapeutic potential of AMP2041.