With the antibiotic development pipeline running dry, many fear that we might soon run out of treatment options. High-density infections are particularly difficult to treat due to their adaptive multidrug-resistance and currently there are no therapies that adequately address this important issue. Here, a large-scale in vivo study was performed to enhance the activity of antibiotics to treat high-density infections caused by multidrug-resistant Gram-positive and Gram-negative bacteria. It was shown that synthetic peptides can be used in conjunction with the antibiotics ciprofloxacin, meropenem, erythromycin, gentamicin, and vancomycin to improve the treatment outcome of murine cutaneous abscesses caused by clinical hard-to-treat pathogens including all ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae) pathogens and Escherichia coli. Promisingly, combination treatment often showed synergistic effects that significantly reduced abscess sizes and/or improved clearance of bacterial isolates from the infection site, regardless of the antibiotic mode of action. In vitro data suggest that the mechanisms of peptide action in vivo include enhancement of antibiotic penetration and potential disruption of the stringent stress response.

There has been enormous publicity about the inexorable rise of resistance and the dearth of new therapies. However less attention has been placed on adaptively multidrug-resistant high density bacterial infections for which antibiotics are highly used but no effective therapies currently exist. Here we have provided new hope for this previously intractable class of infections as typified by abscess infections that are responsible for 3.2 million annual emergency room visits in the US alone. We show how to enhance the activity of antibiotics to treat multidrug-resistant Gram-positive and Gram-negative bacteria, using peptides that target the bacterial stress response, persister-based resistance and the outer membrane permeability barrier. In particular we have employed a new bacterial subcutaneous abscess mouse model to demonstrate that: (a) 7 of the society’s most recalcitrant pathogens formed cutaneous abscesses and even when antibiotics were directly delivered into abscess tissues, they showed poor efficacy; (b) By combining antibiotics with the local administration of anti-biofilm peptides that target cellular (stringent) stress responses, we could pharmacologically treat the infection and reduce the severity of cutaneous abscesses; (c) This synergy was due to increased outer membrane permeability as well as the disruption of the conserved stringent stress response that controls virulence and antibiotic resistance, particularly due to so-called persisters. These peptides have therefore the potential to broaden our limited antibiotic arsenal for a group of extremely difficult to treat infections.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: The peptides described here have been filed for patent protection, assigned to REWH’s employer the University of British Columbia, and licenced to ABT Innovations Inc. in which the University of British Columbia and REWH own shares.

Funding: Research reported in this publication was supported by a grant from the Canadian Institutes for Health Research FDN-154287, the National Institute of Allergy and Infectious Diseases (NIAID) of the U. S. National Institutes of Health under Award Number R33AI098701, and the Intramural Research Program of the NIAID. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. DP received a Feodor Lynen postdoctoral fellowship from the Alexander von Humboldt Foundation (Germany) as well as a Cystic Fibrosis Postdoctoral fellowship (Canada). SCM received the Centre for Blood Research (CBR) graduate student award as well as the Gerhard Henrik Armauer-Hansen Memorial Scholarship. REWH holds a Canada Research Chair in Health and Genomics and a UBC Killam Professorship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

ESKAPE pathogens (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, E. cloacae) are recognized to be responsible for the majority of difficult-to-treat community-acquired, healthcare-associated, and nosocomial infections [ 1 ]. Multidrug-resistant bacteria represent major therapeutic challenges and pose a great threat to human health [ 2 ]. The increasing resistance to available antibiotics dampens treatment possibilities and there is a serious lack of adequate treatment options. Less discussed but of even greater concern are infections associated with high bacterial densities (>10 7 CFU/ml bacteria) especially biofilm and/or abscess infections. High bacterial densities lead to elevated MICs to multiple antibiotics [ 3 ] and are extremely difficult to treat with antibiotics [ 4 ]. In this context, skin and soft tissue infections (SSTIs) are an emerging problem, a significant burden in health care facilities, and responsible for increased antibiotic administration [ 5 ]. SSTIs such as abscesses form fluid, pus-filled pockets infiltrated by bacteria and immune cells [ 6 ], and are often highly resistant to antibiotic treatment. Indeed, abscesses are the most common indication for frequent (6–12 h), high-dose (up to 1 g/kg) and long term (>5 d) [ 7 ] intravenous (IV) broad-spectrum antibiotic administration [ 5 ]. SSTIs have been traditionally thought to be largely caused by S. aureus and Streptococcus pyogenes but recent findings show that other microbes are very prevalent [ 8 , 9 ]. Indeed, the SENTRY antimicrobial surveillance program (North America) [ 10 ] reported that the major pathogens isolated from SSTIs now include 10.8% P. aeruginosa, 8.2% Enterococcus sp., 7.0% E. coli, 5.8% Enterobacter sp., and 5.1% Klebsiella sp., as well as 45.9% S. aureus. Moreover, recently A. baumannii is increasingly recognized as an emerging cause of nosocomial infections and important cause of severe, life-threatening soft tissue infections [ 11 ]. High bacterial numbers of greater than 10 8 CFU/ml isolated bacteria are present in soft-tissue and peritoneal infections [ 12 ], highlighting the importance of investigating high-bacterial density infections. However, standard in vitro susceptibility tests employ modest bacterial concentrations of 2–5 x 10 5 per ml which critically underestimates the strong impact on antibiotic susceptibility of the high concentrations of bacteria in such infections [ 12 ]. Thus, it remains a major challenge to translate in vitro findings into in vivo efficacy and compounds that show excellent in vitro activity (e.g., low MIC in defined medium), often work poorly when tested under in vivo conditions.

Results and discussion

To investigate abscess infections caused by the ESKAPE pathogens and E. coli, we extrapolated from our previously-developed cutaneous mouse infection model [4], prioritizing the study of resistant, recalcitrant host-adapted pathogens rather than commonly used laboratory strains. We identified clinical isolates that were able to cause chronic skin abscesses on the backs of CD-1 female mice after injection of a high bacterial dose (≥ 107 bacteria); each of these strains persisted throughout the course of a three day experiment and did not cause mortality in mice. MIC assays revealed that these strains had generally low antibiotic susceptibility and were resistant to antibiotics from at least three different classes (Table 1, S1 Table). Plasmid-encoded bioluminescently-tagged isolates were created to enable visualization and monitoring of the progress of disease using non-invasive techniques, and to provide evidence that the skin infection contained metabolically active bacteria; this enabled us to follow the infection for all strains (Fig 1).

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larger image TIFF original image Download: Fig 1. In vivo tracking of bioluminescently labeled (live) bacterial infections. CD-1 female mice were injected with individual bacterial strains carrying plasmids constitutively expressing lux reporter genes. Bacterial strains were injected subcutaneously at a dose of 1 × 109 CFU E. faecium, 5 × 107 CFU S. aureus, 1 × 109 CFU K. pneumoniae, 1 × 109 CFU A. baumannii, 5 × 107 CFU P. aeruginosa, 2.5 × 108 CFU E. cloacae, and 1 × 108 CFU E. coli. The infection was monitored 1 h post infection and then every 24 h until day 3. Representative images for day 0 and day 3 are shown. Mice were imaged using a Perkin Elmer in vivo imaging system (IVIS) and the experiment was repeated twice with three mice/group. The scale at the bottom indicates radiance x 106. https://doi.org/10.1371/journal.ppat.1007084.g001

To optimize the treatment strategy, antibiotics were chosen based on their moderate in vitro MIC values (0.02 to 15.6 μg/ml) and empirically tested in vivo to determine an appropriate concentration that reduces abscess sizes and/or CFU just enough to observe synergy of the peptides and antibiotics. Since the drug concentrations after IV injection might be affected by various factors including blood perfusion, penetration into tissues and/or binding to plasma proteins or dermal components [13], we directly injected antibiotics into the infected tissue. This allowed us to overcome the distinct pharmacokinetics of antibiotics used in humans as opposed to mice when applied intravenously, as well as the amounts delivered and time for penetration (S1 Table) to the target site [13, 14]. For each antibiotic directly injected into the infected abscess tissue, an amount that would provide a total body concentration greater than the effective antibiotic dose was chosen. Meropenem was used to treat A. baumannii and K. pneumoniae infections at concentrations of 6 and 10 mg/kg, respectively. The MIC for meropenem against K. pneumoniae was very low (0.1 μg/ml), while the MIC against A. baumannii was quite high (15.6 μg/ml). Based on EUCAST clinical breakpoint information, K. pneumoniae KPLN649 is resistant to meropenem while A. baumannii Ab5075 is sensitive (S1 Table). However intriguingly, treatment of an A. baumannii infection reduced bacterial cell numbers by 111-fold, while there was only a 2.7-fold clearance of K. pneumoniae. Indeed, K. pneumoniae showed the highest recalcitrance towards all tested antibiotic treatments in this skin infection model and high concentrations of azithromycin (500 mg/kg) or colistin (3 mg/kg) had no anti-infective activity. Similarly, although the MICs of ciprofloxacin against P. aeruginosa (3.1 μg/ml) and K. pneumoniae (6.3 μg/ml) were quite similar, and both strains resistant to ciprofloxacin based on EUCAST (S1 Table), as little as 0.4 mg/kg ciprofloxacin were required to reduce the P. aeruginosa load by 15-fold, while a 75-fold greater dosage of 30 mg/kg reduced the K. pneumoniae bacterial burden by only 2-fold. Similarly, the E. coli and E. cloacae strains were sensitive towards ciprofloxacin (S1 Table), with MICs 0.02 and 0.04 μg/ml, respectively; however, in the mouse abscess model 4 mg/kg was required to reduce E. coli cells by 5.8-fold while only 0.006 mg/kg was required to reduce E. cloacae by 2.8-fold (Fig 2A–2E) (S2 Table).

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larger image TIFF original image Download: Fig 2. Antibiotic and synthetic peptide mono- and combinatorial therapy in a murine cutaneous abscess model using female CD-1 mice and clinical drug-resistant bacterial isolates. Bacterial strains were injected subcutaneously and treated one hour post infection with either saline (control), synthetic peptides, antibiotics, or antibiotic-peptide combinations. Synthetic peptide concentrations for all conditions were as follows: 1002, 10 mg/kg (3 mg/kg for E. faecium); 1018, 10 mg/kg; HHC-10 10 mg/kg, and DJK-5, 3 mg/kg (0.25 mg/kg for S. aureus). Infected and inflamed tissue was measured three days post infection and pus-containing abscess lumps excised to determine CFU. Abscess sizes are in box and whiskers plots (left panel) and counted CFU/abscess data expressed with geometric mean (right panel). A. P. aeruginosa LESB58, ciprofloxacin 0.4 mg/kg. B. A. baumannii Ab5075, erythromycin 6 mg/kg, meropenem 6 mg/kg. C. K. pneumoniae KPLN649, meropenem 10 mg/kg, ciprofloxacin 30 mg/kg. D. E. cloacae 218 R1, ciprofloxacin 0.006 mg/kg. E. E. coli E38, ciprofloxacin 4 mg/kg, F. E. faecium #1–1, gentamicin 16 mg/kg. G. S. aureus LAC, clindamycin 0.01 mg/kg; vancomycin 0.15 mg/kg. (A-G) n = 368 biologically independent animals. All experiments were done at least three times with 2–4 mice/group. Statistical analysis was performed using One-way ANOVA, Kruskal-Wallis test with Dunn’s correction (two-sided). The asterisk indicates significant differences to the wild-type (*, p < 0.05; **, p < 0.01; ***, p < 0.001). The hash indicates significant differences of the combination therapy over the sum of the effects of each agent alone (#, p < 0.05; ##, p < 0.01; ###, p < 0.001). https://doi.org/10.1371/journal.ppat.1007084.g002

These important observations highlight that antibiotic mono-therapies as well as high antibiotic dosages are often ineffective when bacteria form high density infections. Thus in vitro MICs are useful indicators of potential, but are not always predictive of in vivo efficacy especially when adaptive resistance occurs. The situation was even more difficult for strains that were resistant towards several classes of antibiotics, e.g. an E. faecium patient isolate that showed extensive drug resistance towards all tested antibiotics (Table 1). In this case the use of gentamicin for in vivo treatment at a high dosage (16 mg/kg) led to a reduction of the bacterial burden by about 4-fold (Fig 2F, S2 Table). Conversely, a methicillin resistant S. aureus infection was somewhat treatable, although sensitive to the used antibiotics based on EUCAST (S1 Table), with clindamycin (0.01 mg/kg) and vancomycin (0.15 mg/kg), both of which visually reduced abscess sizes, but had no impact on bacterial clearance (Fig 2G, S2 Table).

Host defense peptides (HDPs) are small cationic amino acids groups produced by the body as a defense mechanism and are key components of immunity [15], while short synthetic derivatives show promise as broad spectrum anti-infectives that protect in various animal models [16]. A discrete subset of these peptides is effective against a broad spectrum of bacterial biofilms [17]. The mechanism of action of such peptides, e.g. 1018 and DJK-5, has been linked to the disruption of the stringent stress response [17, 18]. The bacterial stringent response is a highly conserved (present in Gram-positive and Gram-negative bacteria) response to various stresses that impacts virulence and antibiotic susceptibility. Their unique ability to disrupt this stress response enables such peptides to show activity against stringent response controlled abscess infections, where biofilm phenotypes have also been suggested to play a crucial role [17, 19, 20]. In this context, although our peptides showed high MICs in vitro (Table 1), we previously showed that they can reduce abscess sizes as well as have modest effects in reducing bacterial numbers when S. aureus or P. aeruginosa infections were treated [18, 20].

Here we hypothesized that they would convert bacteria into a more relaxed (unstressed) state that would render them more susceptible to antibiotic treatment. In this regard, peptide DJK-5 at just 3 mg/kg reduced bacterial loads of P. aeruginosa (4.6-fold), E. faecium (22-fold), K. pneumoniae (4.0-fold), A. baumannii (9.9-fold), and E. coli (2.2-fold) infections (Fig 2A–2C and 2E, S2 Table). Other peptides, namely 1002, 1018, and HHC-10 at concentrations of 10 mg/kg had no significant impact on the bacterial burden but showed promise in visually reducing abscess sizes for P. aeruginosa, E. faecium, K. pneumoniae, E. coli, and E. cloacae infections (Fig 2A, 2C–2F).

Antibiotic combination therapy is frequently used as a possible method of outmaneuvering recalcitrant bacterial pathogens [21] but its application remains controversial and debated, in part due to the increased risk of toxicity, organ damage, and the selection and emergence of resistant strains [22]. Unfortunately, although in vitro assessments of synergy employing checkerboard titration have been used to justify combination therapy, these are rarely followed up with animal model infection studies. Testing synthetic peptides in combination with antibiotics in vivo showed the ability to enhance the treatment outcome of multidrug resistant bacterial infections. In vivo synergy was defined as demonstrating an effect that was significantly more pronounced in the combination than the sum of the effects of each agent alone using saline-treated animals as a negative control [23, 24]. By this definition, combination therapies applied with DJK-5 and antibiotics significantly worked against P. aeruginosa (reduction of bacterial load by 245-fold in combination with ciprofloxacin), E. faecium (265-fold with gentamicin), K. pneumoniae (91-fold with meropenem), A. baumannii (1325-fold with erythromycin and 2006-fold with meropenem), and S. aureus (11-fold with vancomycin) when comparing bacterial burdens to the saline control. Indeed DJK-5 worked synergistically and showed an average overall improvement of 14-fold compared to the sum of the individual single treatments. The highest synergistic activity occurred when DJK-5 was combined with meropenem or erythromycin against A. baumannii, reducing the bacterial load by 65-fold and 33-fold, respectively, in combination treatment compared to the sum of individual treatments (Fig 2B; S2 Table). For E. coli, DJK-5 in combination with ciprofloxacin showed a 10.3-fold reduction in bacterial numbers over the saline control, which was still a 2.9-fold improvement over the summed monotherapies (Fig 2E).

Analogous data was also obtained with other peptides; 1018 in combination with ciprofloxacin showed about 186-fold, 6.9-fold, 5.4-fold, and 8-fold reduction in bacterial burdens against P. aeruginosa, K. pneumoniae, E. cloacae, and E. coli over the saline control (Fig 2A and 2C–2E) and additionally was synergistic in reducing P. aeruginosa and K. pneumoniae numbers from the abscess tissue by 30- and 6.4-fold, respectively compared to the summed monotherapies (Fig 2A and 2C). The combination of HHC-10 with ciprofloxacin against E. cloacae reduced the bacterial load by about 36-fold compared to the saline control and showed synergistic effects over the summed monotherapies (13-fold enhanced activity) (Fig 2D). Gentamicin in combination with peptide 1002 against E. faecium showed an 18-fold reduction in comparison to the control, and a synergistic effect over the sums of the single administrations (7.1-fold increased reduction in bacterial burden) (Fig 2F). Thus, although peptides 1002, 1018, and HHC-10 appeared to be less active in combination treatments, possibly due to their being composed of L-amino acids that makes them susceptible to host proteases, they were also shown to work synergistically under in vivo conditions.

Although genetically-determined antibiotic resistance has been well publicized as a major issue in human health, the current inability to deal with phenotypic multi-drug resistance (e.g. engendered due to growth conditions, especially biofilms), and in particular high-density infections, has not been well addressed. Here, we examined peptides as a possible adjuvant to antibiotic therapy for treating high-density, recalcitrant bacterial abscess infections caused by the most intractable bacterial species. Our strategy to combine conventional antibiotics with synthetic peptides offers a novel therapeutic approach to effectively treat high density infections in that a range of combination therapies were able to reduce the bacterial burden of problematic clinical isolates in our subcutaneous infection model. At least part of the effect of peptides on antibiotic action was likely due to the ability of peptides to elicit degradation of the stringent stress response intracellular signaling molecule ppGpp [17, 25], which has been tied to resistance induction [26, 27], and the development of energy-starved persisters [28, 29]. To further elucidate how the peptides acted, we performed checkerboard titration experiments to determine interactions between the antibiofilm peptides 1018 and DJK-5 and the antibiotic ciprofloxacin against P. aeruginosa LESB58. Since antibiofilm peptides exert their activities under stringent stress conditions [17, 25], such as encountered in biofilms or abscesses, the stringent response (ppGpp) inducing agent serine hydroxamate (SHX) as well as a ppGpp-overproducing strain (LESB58 containing the overexpressed cloned relA gene) were used (S3 Table). As expected, under planktonic growth conditions there was no effect of the combined treatment. However, for the two ppGpp-overexpressing situations, combining the two agents lowered the effective concentrations of each agent to below the MIC, but this was prevented in a stringent response ΔrelAΔspoT double mutant. The insertion of the cloned relA gene into the double mutant enabled effective ciprofloxacin-peptide combinations. In this context, the mutant lacking the stringent response genes, was 2-fold more susceptible towards ciprofloxacin while the mutant complemented with the ppGpp synthetase, RelA, had a 4-fold higher MIC to ciprofloxacin (S3 Table). Stress related responses could provide another mechanism of synergy in addition to peptide-mediated breach of the Gram-negative permeability barrier. These in vitro findings provide a plausible mechanism that may also be occurring in vivo.

Most available antibiotics target intracellular processes and therefore must penetrate the bacterial cell envelope, which is particularly challenging in Gram-negative bacteria due to their formidable outer membrane. To further investigate the molecular basis of the synergistic and additive effects of the combinatorial treatment, we performed outer membrane permeability assays, observing the ability of peptides to enhance the uptake of the normally impermeable hydrophobic fluorophore N-phenyl-1-naphtylamine (NPN). Colistin, a cationic lipopeptide antibiotic that is known to permeabilize the bacterial outer membrane [30], served as a positive control, while meropenem, gentamicin, or vancomycin were used as negative controls. Except for 1002, each peptide was able to interact with and permeabilize the outer membrane of Gram-negative bacteria at their corresponding MICs (Table 2) and/or 10xMICs (S4 Table). The effect of the peptides on Gram-positive bacteria was almost undetectable since NPN has almost unimpeded access to Gram-positive bacteria.

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larger image TIFF original image Download: Table 2. Outer membrane permeabilization by peptides cf. antibiotics at their corresponding MICs. The uptake of the fluorophore NPN in the presence of different antibiotics and synthetic peptides was determined by assessing increased fluorescence at an excitation wavelength of 350 nm and an emission wavelength of 420 nm due to partition of the normally impermeable hydrophobic NPN into bacterial membranes. Relative fluorescence values of at least three biological replicates were determined by subtracting the fluorescence value without test substance. https://doi.org/10.1371/journal.ppat.1007084.t002

The peptides used here worked with a variety of antibiotics. However, future work should explore further possible combinations to find those most optimal. Other possible mechanisms contributing to synergy should be investigated including modulating the host innate immune/inflammatory responses (increasing protective responses while dampening inflammation) and yet-to-be-identified downstream processes associated with the blockage of the stringent response.

The insights from our study could help physicians to understand bacterial infections in skin and soft tissues, and aid in management and development of improved treatment strategies. Ultimately, we have provided evidence that our peptides, especially DJK-5, showed superior effects when paired with antibiotics.