Abstract Increasing multidrug resistance (MDR) in Acinetobacter baumannii warrants therapeutic alternatives, and the bactericidal nature of antimicrobial peptides (AMPs) offers a possible approach. In this study, we examined the interaction of cathelicidin AMPs WAM-1, a marsupial AMP, and LL-37, a human AMP, with A. baumannii clinical isolates. We characterized the antibiotic resistance of the isolates, the bacteriostatic and bactericidal effects of these AMPs, synergistic activity with antibiotics, and their effects on biofilm formation and dispersal. All clinical isolates were resistant to commonly prescribed antibiotics, with four of seven isolates showing MDR. WAM-1 and LL-37 showed variable activity in clinical isolates, with WAM-1 having a stronger bacteriostatic effect than LL-37 and showing rapid bactericidal activity against clinical isolates. Furthermore, synergistic bactericidal activity was observed with WAM-1 and commonly prescribed antibiotics. Both peptides were able to inhibit biofilm formation in all clinical isolates at some concentrations, and WAM-1 dispersed mature biofilm in most isolates. LL-37 was unable to disperse mature biofilms in any strains. Further studies must be done to elucidate the true value of these alternative treatments, but these results suggest that MDR A. baumannii's susceptibility to AMPs may result in innovative therapeutics to prevent or treat these infections. Acinetobacter baumannii, antimicrobial peptides, cathelicidins, antibiotic susceptibility, multidrug resistance, novel therapeutics INTRODUCTION Acinetobacter baumannii is a gram-negative coccobacillus responsible for a significant number of nosocomial infections. As an opportunist, A. baumannii predominantly affects immunocompromised patients (Breslow and Meissler 2011). Common infections include catheter-associated urinary tract infections, ventilator-associated pneumonia, skin and soft tissue infections, and blood stream infections that often lead to septicemia (Howard et al.2012). These infections are easily spread in intensive care units through the formation of biofilms, affecting individuals with suppressed immune systems and patients with wounds acquired from major trauma (Breslow and Meissler 2011). Biofilm formation, the ability to form surfaced-adhered bacterial communities imbedded in a self-produced polymeric matrix, is a virulence factor employed by many pathogens to persist on abiotic surfaces as well as host tissues. Bacteria found within a biofilm are protected from antibiotic treatment as well as the host immune response (Longo, Vuotto and Donelli 2014). Some A. baumannii clinical isolates produce robust biofilms, which aid in nosocomial infections where susceptible immunocompromised patients may have indwelling devices that promote bacterial growth and persistence (Longo, Vuotto and Donelli 2014). Clinical isolates of A. baumannii are often multidrug resistant (MDR), resistant to many disinfectants, and able to survive desiccation, contributing to their persistence (Corbella et al.2000; Poirel and Nordmann 2006; Peleg, Seifert and Paterson 2008). Furthermore, biofilm formation is often observed in clinical strains and positively correlates with drug resistance, likely by providing physical protection that supplements intrinsic resistance mechanisms (Gurung et al.2013; Zarrilli 2016). Widespread MDR and the rapidly declining number of therapeutic options for bacterial ‘superbugs’ necessitates the development of novel antimicrobials. One alternative to traditional antibiotics is the use of cathelicidin antimicrobial peptides (AMPs) similar to those found in the innate immune system (Stewart 2002; Poirel and Nordmann 2006; Wang et al.2011). AMPs are an evolutionarily conserved, heterogeneous group of short oligopeptides produced by the innate immune system and shown to have broad-spectrum bactericidal activity against pathogens including viruses, bacteria and parasites, serving as an integral part of the immune system's first line of defense (Poirel and Nordmann 2006; Bahar and Ren 2013; Galdiero et al.2015). Numerous AMPs have been isolated from natural sources and many others have been synthetically produced (Bahar and Ren 2013; Galdiero et al.2015). They demonstrate antimicrobial activity in the micromolar range and, compared with traditional antibiotics, kill bacteria very rapidly (Galdiero et al.2015; Dutta and Das 2016). Cathelicidins are a well-known family of AMPs known for their effectiveness against a wide range of bacteria, including A. baumannii (Wang et al.2011). Previous studies have shown that LL-37, the only human cathelicidin, disables bacteria through membrane disruption due to non-specific peptide–lipid interactions, exhibiting bactericidal activity against both gram-positive and gram-negative bacteria at micromolar concentrations. In the presence of human serum, however, the antibacterial activities of LL-37 are diminished, making this an unlikely therapeutic in target tissues such as the lung or blood (Johansson et al.1998; Turner et al.1998; Feng et al.2013). Research is currently focused on many natural and synthetic AMPs that may have therapeutic value either alone or in combination with existing antibiotics (Gopal et al.2014). The marsupial AMP WAM-1 is one of 14 cathelicidins found in the tammar wallaby and although the mechanism of action has yet to be determined, WAM-1 has been shown to be 12 to 80 times more effective than LL-37 in its ability to kill several bacterial pathogens, including several clinical isolates of A. baumannii (Wang et al.2011). Unlike LL-37, it is resistant to inhibition by high salt concentrations and is also non-hemolytic, indicating that it may be suitable for applications in vivo (Wang et al.2011; Dutta and Das 2016; Cheng and Belov 2017). One of the therapeutic difficulties with AMPs is their loss of function in target tissues, but WAM-1 may not face these limitations (Wang et al.2011). This suggests that WAM-1 has the potential to be an effective alternative to traditional antibiotic therapy. The development of microbial resistance against naturally occurring AMPs is rare, making WAM-1 a good candidate in the search for natural peptides to fight MDR pathogens (Wang et al.2011). The aim of this study was to elucidate the effects of LL-37 and the marsupial cathelicidin WAM-1 on a collection of clinical isolates including several strains of MDR A. baumannii. We characterized the susceptibility of clinical isolates to commonly used antibiotics, the innate cathelicidin LL-37, and the marsupial cathelicidin WAM-1. We also explored the bactericidal effect and synergistic activity between WAM-1 and commonly prescribed antibiotics. Furthermore, we examined LL-37 and WAM-1’s ability to inhibit biofilm formation and to disperse mature biofilms. MATERIALS AND METHODS Bacterial strains and growth conditions Clinical isolates of A. baumannii, designated LK10, LK14, LK15, LK41, LK49, LK80 and LK88, were obtained from the G. V. Sonny Montgomery Veteran's hospital clinical laboratory in Jackson, MS. Bacteria were stored at –80°C in Luria-Burtani (LB) broth supplemented with 20% glycerol. The isolates were plated on MacConkey agar (BD) and grown overnight at 37°C for use in further experiments. Minimum inhibitory concentrations The minimum inhibitory concentration (MIC) of ciprofloxacin, imipenem, and amikacin were determined by the gradient diffusion method using E-test strips on Mueller-Hinton (MH) agar (bioMerieux, Marcy l’Etoile, France). Clinical isolates were grown to mid-log phase (OD600 = 0.5) in MH broth by shaking at 37°C for ∼2 h. A sterile cotton swab was saturated with the inoculum and used to evenly streak the surface of a MH agar plate in accordance with manufacturer's instructions. Plates were incubated for 24 h in an aerobic atmosphere at 37°C. The antibiotic concentration range of the E-test was 0.016–256 μg ml−1, and MIC values were read at the point of 80% inhibition (Wiegand, Hilpert and Hancock 2008). AMP LL-37 was purchased from AnaSpec Inc, and WAM-1 was synthesized by Thermo Fisher Scientific (WAM-1 = KRGFGKKLRKRLKKFRNSIKKRLKNFNVVIPIPLPG). MIC values of each were measured using the microbroth dilution method in MH broth according to Clinical and Laboratory Standards Institute standards (Wiegand, Hilpert and Hancock 2008). Each peptide was serially diluted into 50 μl of 0.01% acetic acid to yield concentrations of 250, 166.7, 111.1, 74.1, 49.4, 32.9, 21.9 and 14.6 μg ml−1. A negative control was performed by incubating the inoculum with 50 μl of 0.01% acetic acid containing no peptide. Clinical isolates LK 10, 14, 15, 41, 49, 80 and 88 were grown to mid-log phase (OD600 = 0.5) in MH broth by shaking at 37°C for 2 h. After adding 100 μl of inoculum to the diluted peptide, samples were then incubated at 37°C for 18 h. Following incubation, absorbance was read at 600 nm using a Bio-Rad microplate absorbance reader. Each assay was done in triplicate with three biological replicates. Survival assays The bactericidal effects of antibiotics and AMPs were characterized as previously described with modifications using a killing-curve technique (Giamarellou and Petrikkos 1987). Three representative MDR isolates, LK10, LK15 and LK49, were grown to mid-log phase (OD600 = 0.5) in MH broth. The following six treatments were done as previously described to assess synergistic activity with each antibiotic (Acros Organics) and WAM-1: untreated control (no AMP, no antibiotic), antibiotic alone (10 μg ml−1), 0.5X MIC of WAM-1 (125 μg ml−1), 0.5X MIC WAM-1 (125 μg ml−1) + antibiotic (10 μg ml−1), 2X MIC of WAM-1 (500 μg ml−1) and 2X MIC WAM-1 (500 μg ml−1) + antibiotic (10 μg ml−1) (Wang et al.2011). Each sample contained 1 ml of inoculum, 1 ml of antibiotic (ciprofloxacin, amikacin or imipenem) diluted to a working concentration in 1X phosphate-buffered saline (PBS) and 200 μl of peptide diluted into 0.01% acetic acid to the appropriate concentration. Samples were then incubated for 0, 15, 60, 120 and 180 min in an aerobic atmosphere at 37°C. At each time point, samples were serially diluted in PBS and plated for viability onto MacConkey agar. Colonies were counted after 24 h of incubation at 37°C. This was repeated for each strain. Biofilm assay As previously described, clinical isolates of A. baumannii were grown to mid-log phase and diluted 1:1 with sterile LB broth in the wells of a 96-well microtiter plate to a final volume of 100 μl (King et al.2009; Gurung et al.2013; King Pangburn and McDaniel 2013). Treatments with LL-37 or WAM-1 were done at the following concentrations: 250, 166.7, 111.1, 74.1, 49.4, 32.9, 21.9 and 14.6 μg ml−1. The negative control was treated with 10 μl of 0.01% acetic acid. For pre-treatment assays, 10 μl of the correct peptide treatment was added at the time of inoculation and plates were incubated for ∼16 h at 37°C. For post-treatment assays, samples were incubated overnight in the absence of peptide treatment to allow mature biofilms to form, treated with 10 μl of the appropriate AMP at 16 h and incubated for an additional 24 hours. Following the final incubation, wells were washed four times with distilled water and stained with 0.1% crystal violet for 30 min at room temperature. Wells were washed four times with distilled water and trapped crystal violet eluted with 200 μl of 95% ethanol. This eluent (125 μl) was transferred to a fresh plate and the absorbance read at 575 nm using a Bio-Rad microplate reader. Statistics All experiments were performed in triplicate a minimum of three times independently. Statistical analyses were performed using JMP (Version 9, SAS Institute Inc., Cary, NC). Comparisons between treated and untreated groups were performed using the Mann-Whitney two-sample rank test. P values of <0.05 were considered statistically significant. RESULTS MDR A. baumannii strains are resistant to LL-37 but sensitive to WAM-1 MICs were determined for three antibiotics and each strain classified as sensitive, intermediate, or resistant to each antibiotic (Table 1). The drugs chosen represent three of the most commonly prescribed drugs in the treatment of A. baumannii, three different classes of antibiotics, and different mechanisms of action. As previously observed, several of these clinical isolates were resistant to all three classes of antibiotics (Peleg, Seifert and Paterson 2008; King et al.2009; Howard et al.2012). Five of the seven isolates were resistant to one or more class of antibiotics (resistance defined as MIC > 4), and four isolates, LK10, LK14, LK15, and LK49, were MDR. MIC assays performed with each of the cathelicidin AMPs demonstrated that the marsupial peptide WAM-1 had a bacteriostatic effect on all clinical isolates, with an MIC of ≤250 μg ml−1 (Table 2). Innate human AMP LL-37, however, was not effective at inhibiting growth and MIC values were >250 μg ml−1 for all isolates. While both of these AMPs are cathelicidins, published sequences show significant deviation with only six amino acid residues conserved, indicating possible functional differences (Fig. 1) (Wang et al.2011; Luo et al.2017). Figure 1. View largeDownload slide Amino acid sequences of cathelicidins WAM-1 and LL-37 differ significantly, potentially explaining differences in activity. Sequences were obtained from previously published studies and aligned to elucidate similarities and differences at the amino acid level. Figure 1. View largeDownload slide Amino acid sequences of cathelicidins WAM-1 and LL-37 differ significantly, potentially explaining differences in activity. Sequences were obtained from previously published studies and aligned to elucidate similarities and differences at the amino acid level. Table 1. MICs of ciprofloxacin, imipenem, and amikacin in clinical strains of A. baumannii. Isolate Antibiotic MIC (μg ml−1) Sensitivity LK10 Amikacin >256 R Imipenem 32 R Ciprofloxacin 32 R LK14 Amikacin 6 R Imipenem 24 R Ciprofloxacin 32 R LK15 Amikacin 4 I Imipenem 16 R Ciprofloxacin 32 R LK41 Amikacin 2 R Imipenem 0.19 S Ciprofloxacin 0.25 S LK49 Amikacin >256 R Imipenem 32 R Ciprofloxacin 32 R LK80 Amikacin 1 S Imipenem 0.19 S Ciprofloxacin 0.17 S LK88 Amikacin 4 I Imipenem 0.19 S Ciprofloxacin 0.10 S Isolate Antibiotic MIC (μg ml−1) Sensitivity LK10 Amikacin >256 R Imipenem 32 R Ciprofloxacin 32 R LK14 Amikacin 6 R Imipenem 24 R Ciprofloxacin 32 R LK15 Amikacin 4 I Imipenem 16 R Ciprofloxacin 32 R LK41 Amikacin 2 R Imipenem 0.19 S Ciprofloxacin 0.25 S LK49 Amikacin >256 R Imipenem 32 R Ciprofloxacin 32 R LK80 Amikacin 1 S Imipenem 0.19 S Ciprofloxacin 0.17 S LK88 Amikacin 4 I Imipenem 0.19 S Ciprofloxacin 0.10 S S, sensitive; I, intermediate; R, resistant. View Large Table 2. MICs of cathelicidin AMPs LL-37 and WAM-1 in clinical strains of A. baumannii. Isolate AMP MIC (μg ml−1) LK10* LL-37 >250 WAM-1 250 LK14* LL-37 >250 WAM-1 250 LK15* LL-37 >250 WAM-1 166 LK41 LL-37 >250 WAM-1 166 LK49* LL-37 >250 WAM-1 250 LK80 LL-37 >250 WAM-1 166 LK88 LL-37 >250 WAM-1 166 Isolate AMP MIC (μg ml−1) LK10* LL-37 >250 WAM-1 250 LK14* LL-37 >250 WAM-1 250 LK15* LL-37 >250 WAM-1 166 LK41 LL-37 >250 WAM-1 166 LK49* LL-37 >250 WAM-1 250 LK80 LL-37 >250 WAM-1 166 LK88 LL-37 >250 WAM-1 166 *Denotes MDR strains. View Large WAM-1 acts synergistically with antibiotics Three clinical isolates, LK10, LK15, and LK49, were chosen as representative MDR strains and used in time-course survival assays. Consistent with MIC data (Table 1), all three strains survived a 3-h treatment with antibiotic alone, demonstrating survival was not statistically different than untreated controls (Fig. 2). One representative strain, LK10, is shown but effects were consistent for all MDR strains tested. WAM-1 showed rapid bactericidal activity against MDR isolates, decreasing the enumerable viable bacteria by 90% in the first 15 min of treatment. When A. baumannii was treated with 0.5X the MIC of WAM-1 and each of the three antibiotics that it was resistant to, synergistic activity was observed, which is indicated by a significant decrease in cell viability compared to WAM-1 at 0.5X MIC alone (Fig. 2). Figure 2. View large Download slide WAM-1 rapidly kills A. baumannii and has synergistic activity with commonly prescribed antibiotics. MDR A. baumannii was treated with ciprofloxacin (A), amikacin (B) and imipenem (C) alone or in combination with WAM-1, where indicates untreated control, indicates antibiotic treatment alone, indicates treatment with 0.5X MIC of WAM-1, indicates treatment with 2X MIC of WAM-1, indicates treatment with 0.5X the MIC of WAM-1 in combination with each antibiotic, and indicates treatment with 2X the MIC of WAM-1 and each antibiotic. Data for representative strain LK10 are shown, but all three strains tested had consistent results. Plotted points indicate mean cfu ml−1 ± SEM. Figure 2. View large Download slide WAM-1 rapidly kills A. baumannii and has synergistic activity with commonly prescribed antibiotics. MDR A. baumannii was treated with ciprofloxacin (A), amikacin (B) and imipenem (C) alone or in combination with WAM-1, where indicates untreated control, indicates antibiotic treatment alone, indicates treatment with 0.5X MIC of WAM-1, indicates treatment with 2X MIC of WAM-1, indicates treatment with 0.5X the MIC of WAM-1 in combination with each antibiotic, and indicates treatment with 2X the MIC of WAM-1 and each antibiotic. Data for representative strain LK10 are shown, but all three strains tested had consistent results. Plotted points indicate mean cfu ml−1 ± SEM. WAM-1 effectively inhibits and disperses biofilm while LL-37 only inhibits its formation To examine the ability of these cathelicidins to inhibit or disperse biofilms produced by A. baumannii, a quantitative crystal violet assay was used to examine all clinical isolates in our collection; representative MDR strains are shown in Figure 3 and all data are summarized in Table 3. Treatments were administered either at the time of inoculation (termed pre-treatment) or after 16 h of incubation (termed post-treatment) to characterize either inhibition or dispersal. Treatment with LL-37 inhibited biofilm formation in all seven clinical isolates but was unable to disperse mature biofilm in any strain (Fig. 3A and C, Table 3). Treatment with WAM-1 inhibited biofilm formation in all seven strains and dispersed biofilm in six of seven isolates, including three of the four MDR strains (Fig. 3B and C, Table 3). In strains that were effectively treated by one of the AMPs, these results were often not dose-dependent. Overall, WAM-1 was more effective than LL-37 at dispersal of mature biofilms, but both peptides effectively inhibited biofilm formation in all strains. Figure 3. View largeDownload slide WAM-1 and LL-37 inhibit biofilm formation in MDR A. baumannii, but LL-37 fails to disperse mature biofilms. Clinical isolates were treated with increasing concentrations of AMP before or after biofilm formation, incubated overnight and stained with crystal violet to determine the AMP’s ability to inhibit biofilm formation (A, B) or disperse mature biofilms (C, D). Three representative MDR isolates are shown. Biofilms were quantified and graphs indicate average (± SEM) absorbance at 575nm. Asterisks indicate samples that were significantly different from the untreated (P-values < 0.05). Figure 3. View largeDownload slide WAM-1 and LL-37 inhibit biofilm formation in MDR A. baumannii, but LL-37 fails to disperse mature biofilms. Clinical isolates were treated with increasing concentrations of AMP before or after biofilm formation, incubated overnight and stained with crystal violet to determine the AMP’s ability to inhibit biofilm formation (A, B) or disperse mature biofilms (C, D). Three representative MDR isolates are shown. Biofilms were quantified and graphs indicate average (± SEM) absorbance at 575nm. Asterisks indicate samples that were significantly different from the untreated (P-values < 0.05). Table 3. Summary of the anti-biofilm activity of cathelicidin AMPs LL-37 and WAM-1 in clinical strains of A. baumannii. Isolate AMP Biofilm Inhibitiona Biofilm Dispersala LK10* LL-37 Yes No WAM-1 Yes Yes LK14* LL-37 Yes No WAM-1 Yes Yes LK15* LL-37 Yes No WAM-1 Yes Yes LK41 LL-37 Yes No WAM-1 Yes Yes LK49* LL-37 Yes No WAM-1 Yes No LK80 LL-37 Yes No WAM-1 Yes Yes LK88 LL-37 Yes No WAM-1 Yes Yes Isolate AMP Biofilm Inhibitiona Biofilm Dispersala LK10* LL-37 Yes No WAM-1 Yes Yes LK14* LL-37 Yes No WAM-1 Yes Yes LK15* LL-37 Yes No WAM-1 Yes Yes LK41 LL-37 Yes No WAM-1 Yes Yes LK49* LL-37 Yes No WAM-1 Yes No LK80 LL-37 Yes No WAM-1 Yes Yes LK88 LL-37 Yes No WAM-1 Yes Yes a Effective inhibition or dispersal defined as treatments that had significantly less (P value < 0.05) biofilm than untreated controls. *Denotes MDR strains. View Large DISCUSSION Acinetobacter baumannii is highly competent, and resistance genes from other genera such as Salmonella and Shigella have been found within its genome, particularly within a large pathogenicity island (Fournier et al. 2006; Howard et al.2012). MDR A. baumannii poses a distinct threat to immunocompromised patients in clinical settings, prompting a necessary response from researchers investigating novel therapies that do not promote resistance while improving patient outcomes (Corbella et al.2000; Peleg, Seifert and Paterson 2008; Howard et al.2012). With the current lack of viable therapeutic options for these MDR infections, investigations into AMPs have become increasingly widespread (Peleg, Seifert and Paterson 2008; Wang et al.2011). AMPs are promising options, as bacteria do not develop resistance to them (Peleg, Seifert and Paterson 2008; Howard et al.2012). The marsupial cathelicidin peptide WAM-1 exhibits promising evidence of bactericidal properties and the ability to maintain activity in high salinity environments without causing hemolysis, suggesting its possible non-toxic therapeutic use (Wang et al.2011,; Dutta and Das 2016; Cheng and Belov 2017). Our study confirmed consistent inhibitory activity of WAM-1 against seven clinical isolates of A. baumannii, including several MDR strains (Tables 1 and 2), consistent with previous studies (Wang et al.2011). Contrary to previously published studies, however, this antibacterial effect was not observed for the innate cathelicidin LL-37 (Table 2) (Johansson et al.1998; Feng et al.2013). Interestingly, in ongoing work looking at intracellular survival of A. baumannii, we have observed that several strains are able to survive phagocytosis by human neutrophils (data not shown). The resistance to LL-37 demonstrated in this study may offer a potential explanation for intracellular survival of A. baumannii and subsequent immune evasion and infection. The role of LL-37 in the phagocytic killing of A. baumannii has yet to be elucidated and is an important area of ongoing research. Due to WAM-1’s consistent inhibitory effects, we next sought to test its bactericidal activity as well as the efficacy of combining WAM-1 with commonly prescribed antibiotics that MDR strains have developed resistance to. For these experiments, we used previously described clinical isolates resistant to three or more classes of antibiotics (King et al.2009). Bacteria were treated with WAM-1, each antibiotic alone, or a combination of the antibiotic and WAM-1 at either 0.5X the MIC or 2X the MIC. Cultures were incubated for 3 h with the appropriate treatment and plated for viability. In MDR isolates, WAM-1 showed rapid bactericidal activity in as little as 15 min (Fig. 2). Furthermore, WAM-1 showed statistically significant synergistic effects with each of the tested antibiotics, even at 0.5X the MIC of WAM-1. It is interesting that WAM-1 displays synergism with each of the different antibiotics tested, as they represent three different classes and differing mechanisms. The mechanism of action for WAM-1 has yet to be elucidated, but it is a member of the cathelicidin family and likely associates with the bacterial cell membrane through electrostatic interactions as seen in other cathelicidins. The peptide sequence of WAM-1, however, differs significantly from LL-37 which may account for the differences in activity observed as well as the differences in toxicity previously documented (Fig. 1) (Wang et al.2011). This could suggest a different mechanism from that of other cathelicidins. Because the precise mechanism is unknown as well as WAM-1’s interaction with antibiotics, we cannot be certain of the mechanism of synergy. It is possible that WAM-1, like other cathelicidins, disrupts the integrity of the cell membrane which may help overcome some resistance mechanisms and facilitate or enhance the action of many classes of antibiotics. It is also possible that WAM-1 may interact with antibiotics in complementary ways or associate to form a compound with more potent antimicrobial activity than either treatment alone. Considering the dramatic bactericidal effects of WAM-1, its synergistic activity with antibiotics that were of no prior use due to bacterial resistance, as well as its lack of cytotoxicity, WAM-1 represents a viable therapeutic alternative to traditional antibiotic therapy (Wang et al.2011; Dutta and Das 2016; Cheng and Belov 2017). With the increasing level of drug resistance seen in A. baumannii and the ever-increasing number of antibiotics that cannot be used to treat these infections, this discovery may improve treatment outcomes. The formation of robust biofilms, particularly on indwelling devices, represents one of the most important virulence factors involved in nosocomial occurrences of A. baumannii (Long et al.2014; Zarrilli 2016). We demonstrated that both peptides effectively inhibited biofilm formation at some concentrations, but this effect was not dose-dependent and showed inconsistencies between different concentrations of peptide (Fig. 3). WAM-1 effectively dispersed 24-h biofilms in six of the seven isolates tested, including MDR strains LK10, LK14 and LK15, while LL-37 was unable to disperse mature biofilms in any of the strains. Notably, LK80 and LK88 have been previously characterized as high biofilm-producing strains and both were effectively inhibited by both peptides and dispersed by WAM-1 (King et al.2009). These encouraging data suggest that both peptides could be useful in preventing biofilm formation and that WAM-1 could be important therapeutically. Overall, WAM-1 was more effective at inhibiting and dispersing biofilms in our clinical isolates, including MDR strains (Fig. 3 and Table 3). As a therapeutic alternative, WAM-1 in particular may represent a more viable candidate than LL-37 in terms of bactericidal action, synergism with antibiotics, as well as affecting A. baumannii’s ability to form biofilms. This has promising implications. Further studies must be done to reveal the true value of these alternative treatments, but understanding A. baumannii’s susceptibility to AMPs may result in novel therapeutic approaches in the treatment and prevention of MDR infections caused by this important pathogen. Acknowledgements The authors gratefully acknowledge John Barone, Kevin Burgess, and Scott Silvis (Columbus State University) for assistance with statistical analysis and Larry McDaniel (University of Mississippi Medical Center) for kindly providing A. baumannii clinical isolates. FUNDING This work was supported by Columbus State University in the form of a University Grant, multiple Student Research and Creative Endeavors Grants, and funding from the College of Letters and Sciences and the Department of Biology. Further support was provided by the βββ biological honor society and the Flora Clark Research Foundation. Conflicts of interest. None declared. REFERENCES Bahar A, Ren D. Antimicrobial peptides. Pharmaceuticals 2013; 6: 1543– 75. Google Scholar CrossRef Search ADS PubMed Breslow J, Meissler Jr. Innate immune responses to systemic Acinetobacter baumannii infection in mice: neutrophils, but not interleukin-17, mediate host resistance. Infect Immun 2011; 79: 3317– 27. Google Scholar CrossRef Search ADS PubMed Cheng Y, Belov K. Antimicrobial protection of marsupial pouch young. Front Microbiol 2017, DOI: 10.3389/fmicb.2017.00354. Corbella X, Montero A, Pujol M et al. 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Pathogens and Disease – Oxford University Press
Published: Mar 1, 2018
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