Microbial Pathogenesis 142 (2020) 104050

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Combinations of early generation antibiotics and antimicrobial peptides are T

effective against a broad spectrum of bacterial biothreat agents
Christopher K. Cotea,∗, Irma I. Blancoa, Melissa Huntera, Jennifer L. Shoea,
Christopher P. Klimkoa, Rekha G. Panchalb, Susan L. Welkosa,∗∗
a
Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, Frederick, MD, 21702-5011,
USA
b
Countermeasures Division, USAMRIID, Fort Detrick, MD, USA

ARTICLE INFO ABSTRACT

Keywords: The misuse of infectious disease pathogens as agents of deliberate attack on civilians and military personnel is a
Antimicrobial peptides serious national security concern, which is exacerbated by the emergence of natural or genetically engineered
Antibiotics multidrug resistant strains. In this study, the therapeutic potential of combinations of an antibiotic and a broad-
Resistance spectrum antimicrobial peptide (AMP) was evaluated against five bacterial biothreats, the etiologic agents of
Bioterror agents
glanders (Burkholderia mallei), melioidosis (Burkholderia pseudomallei), plague (Yersinia pestis), tularemia
Therapeutics
(Francisella tularensis), and anthrax (Bacillus anthracis). The therapeutics included licensed early generation
Combinations
antibiotics which are now rarely used. Three antibiotics and one 24- amino acid AMP were selected based on
MIC assay data. Combinations of the AMP and tigecycline, minocycline, or novobiocin were screened for sy-
nergistic activity by checkerboard MIC assay. The combinations each enhanced the susceptibility of several
strains. The tetracycline-peptide combinations increased the sensitivities of Y. pestis, F. tularensis, B. anthracis and
B. pseudomallei, and the novobiocin-AMP combination augmented the sensitivity of all five. In time-kill assays,
down-selected combinations of the peptide and minocycline or tigecycline enhanced killing of B. anthracis, Y.
pestis, F. tularensis, and Burkholderia mallei but not B. pseudomallei. The novobiocin-AMP pair significantly re-
duced viability of all strains except B. mallei, which was very sensitive to the antibiotic alone. The results
suggested that antibiotic-AMP combinations are useful tools for combating diverse pathogens. Future studies
employing cell culture and animal models will utilize virulent strains of the agents to investigate the in vivo
availability, host cytotoxicity, and protective efficacy of these therapeutics.

1. Introduction especially hazardous and resistant bacteria. These therapeutic options

The emergence of bacteria which are resistant to antibiotics has
been an increasing problem in treating bacterial infections. This is an
especially critical concern for biothreat pathogens as some of these
strains already have high natural resistance to a range of antimicrobials
(e.g., the Burkholderia). Furthermore, the development of multiple an-
tibiotic resistant (AMR) remains a possible catastrophic scenario. Thus,
the development of new antimicrobial strategies is an urgent need. In
one strategy, previously characterized but overlooked antimicrobials
could be repurposed as potentially successful new treatments for
would include early generation licensed antibiotics which are now
rarely used due to their perceived excessive toxicity and limited ther-
apeutic window.
A second strategy utilizes antimicrobial peptides (AMPs), a diverse
class of antimicrobials which are naturally occurring or synthetically
produced, are 12–50 amino acids in length, and exhibit broad-spectrum
activity [1,2]. Although bacterial resistance to AMPs has been reported,
it is considered to be rare [2,3]. Thus AMPs are promising non-anti-
biotic accessory therapeutics [1,2,4]. They include well-studied natu-
rally occurring AMPs, e.g., cecropin A, LL-37, magainin II, mastoparan,

Abbreviations: AMP, antimicrobial peptide; Bm, Burkholderia mallei; Bp, Burkholderia pseudomallei; Yp, Y. pestis; Ft, F. tularensis; Ba, B. anthracis; Ec, Escherichia coli;
Ps, Pseudomonas aeruginosa; LBG, Luria-Bertani medium with glycerol; MIC, antimicrobial minimal inhibitory concentration; MBC, minimum bactericidal con-
centration; FICI, Fractional Inhibitory Concentration Index; combo, combination; AMR, antimicrobial resistant; combo, combination
∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: christopher.k.cote.civ@mail.mil (C.K. Cote), susan.l.welkos.vol@mail.mil (S.L. Welkos).

https://doi.org/10.1016/j.micpath.2020.104050
Received 4 December 2019; Received in revised form 6 February 2020; Accepted 7 February 2020
Available online 09 February 2020
0882-4010/ Published by Elsevier Ltd.
C.K. Cote, et al.
and melittin [5–13]. Also, synthetic peptides have been engineered
based on natural AMPs. For example, the 12 amino acid cationic pep-
tide bactenecin inhibits growth of some gram-negative bacteria. Wu
et al. [14] constructed several bactenecin analogs that exhibited in-
creased inhibitory activity for both gram-negative and gram-positive
bacteria [14,15]. Hybrid AMPs have also been designed based on ce-
cropin A, LL-37, and magainin II and have been shown to provide
broad-spectrum antimicrobial activity [9]. AMPs have long been of
interest as potentially clinically relevant therapeutics. Several limita-
tions have delayed their successful clinical use. Natural AMPs can be
inhibited by their sensitivity to adverse host-like conditions (e.g., acidic
pH, salts, protease digestion); and nonspecific binding to off-target host
tissues and serum proteins. Also, their use is hampered by potential host
toxicity [1,2,16,17]. However, these limitations may be significantly
ameliorated by use of engineered peptides such as WLBU2, a cationic
helical peptide, which was optimized in length and amino acid content
for antibacterial activity. It was reported to have in vitro activity against
three gram-negative biothreat strains; kill intracellular pathogens in
mouse and human cells; and prevent biofilm development on epithelial
cells, with little or no cytotoxicity [4,18–20]. Finally, bacteriocins re-
present another class of AMPs that could be repurposed to treat bio-
threat agents. They specifically destabilize the plasma membranes of
bacteria, causing cell lysis, and thus are widely regarded to be safe for
humans [21,22].
Naghmouchi et al. [23] showed that there are synergistic effects
between the early generation antibiotic colistin and bacteriocin when
used in combination against gram-negative pathogens such as Pseudo-
monas aeruginosa (Pa). Until recently, colistin had fallen out of favor
and was only used as a last resort for AMR gram-negative bacteria
because of its potential nephrotoxicity. However, when colistin was
combined with a bacteriocin the efficacy of both increased, thus low-
ering their effective concentrations and reducing toxicity of the com-
bination for mammalian cells [23]. Since resistance to colistin has re-
cently emerged, the reduction in the antibiotic's effective dose afforded
by a synergistic therapeutic combination is especially vital.
B. pseudomallei, the agent of melioidosis, and B. mallei, responsible
for glanders, are considered to be potential biological warfare or bio-
terrorism agents and are listed as a Tier 1 biological select agent by the
US Department of Health and Human Services [24] (www.selectagents.
gov/selectagentsandtoxinslist.html). They are intrinsically resistant to
many antibiotics, including beta-lactam antibiotics, aminoglycosides,
macrolides, and polymixins such as colistin. However other older,
rarely used antimicrobials besides colistin are available to potentially
counter drug resistant bacteria such as the Burkholderia, e.g., fosfo-
mycin, nitrofurantoin, tigecycline, minocycline, fusidic acid, and others
[25–30]. For instance, novobiocin is a narrow-spectrum antibiotic
considered to be primarily effective against gram-positive pathogens
but less effective for treating infections caused by gram-negative bac-
teria due to reduced permeability or their outer membrane. However
evidence supports the use of various small molecules which can
permeate membranes to facilitate the concomitant entry of antibiotics
such as novobiocin [30,31]. Furthermore, these antibiotic adjuncts may
effectively reduce the antibiotic minimal inhibitory concentration
(MIC), and we hypothesize that a similar phenomenon could occur by
combining an AMP with an antibiotic.
Table 1
Panel of surrogate bacterial strains used.
Bacterial strain (abbreviation)a Characteristics
B. anthracis Sterne (Ba) capsule-negative strain; animal vaccine
Y. pestis pgm− pPst− (Yp) pgm- pPst- mutant of Yp strain CO92
B. pseudomallei JW270 (Bp) capsule-negative mutant of Bp strain DD503
B. mallei CLH001 ΔtonB, Δhcp1 (Bm) siderophore and virulence gene deletion mutant of Bm ATCC23344
F. tularensis subsp holartica LVS (Ft) spontaneous attenuated derivative; live vaccine strain
a
Highly attenuated and select agent-excluded strains derived from prototype select agent pathogenic strains.
2
Microbial Pathogenesis 142 (2020) 104050
In summary, the presence of AMR strains of biothreat bacteria can
potentially limit the availability of effective therapeutics. Treatment
options may be greatly limited, especially with Burkholderia infections
of which recurrent infections are also a significant problem. Thus, the
development of new antimicrobial therapies, such as the proposed
combination strategy, is critically important. Furthermore, as shown for
other novel therapeutic candidates, the proposed combination ap-
proach may allow the development of more flexible, patient- and in-
fection-specific options for customizing treatment.
The goal of our studies is to identify antibiotics which, when com-
bined with an AMP, exhibit enhanced antibacterial activity against a
broad spectrum of bacterial biothreats and their AMR variants. The
antibiotics selected included licensed, early generation antibiotics
which are currently rarely used and have the potential to be effective
against five bacterial biothreat agents: the etiologic agents of glanders
(Burkholderia mallei), melioidosis (Burkholderia pseudomallei), plague
(Yersinia pestis), tularemia (Francisella tularensis), and anthrax (Bacillus
anthracis). Due to their extensive resistance to many AMPs and innate
antibiotic resistance, the Burkholderia spp. were used as the “bench-
mark” for assessing AMP and antibiotic sensitivity. In future work,
down-selected antibiotic-AMP combinations with synergistic or en-
hanced in vitro activity will be evaluated for their pharmacokinetic
properties, safety, and therapeutic efficacy in animal models.
2. Materials and Methods
2.1. Bacterial strains, media, and growth conditions
The bacterial strains used included five exempt select-agent strains
derived from the etiologic agents of glanders, melioidosis, plague, tu-
laremia, and anthrax: B. mallei (Bm), B. pseudomallei (Bp), Y. pestis (Yp),
F. tularensis (Ft), and B. anthracis (Ba), respectively. These strains are
attenuated mutants derived from a wild type prototype strain as de-
scribed in Table 1 [32–37]. Suspensions of the bacteria were freshly
prepared for assays using strain stocks maintained at −80 °C. Strains Bp
JW270 and E. coli (Ec) ATCC 25922 (quality control strain) were cul-
tured on sheep blood agar plates (SBAP, Thermo Fisher-Remel) and
incubated at 37 °C for 24 h. Bm CLH001 was grown on Luria-Bertani
medium (Lennox formulation) with 4% glycerol (LBG) agar plates
supplemented with 200 μM iron sulfate and incubated at 37 °C for 48 h
[32]. Yp pgm- pPst− was streaked on an SBAP and grown for 48 h at
28–30 °C. Ft LVS was cultured on a chocolate agar (CA) plate (Thermo
Fisher-Remel) for 48 h at 37 °C, colonies were suspended in cation-
adjusted Mueller-Hinton Broth (MHB) (BBL™, BD Diagnostics Franklin
Lake, NJ), and the suspension used to inoculate a flask with 10 ml MHB
supplemented with 2% IsovitaleX (BBL™, BD Diagnostics) from a freshly
rehydrated vial. The broth culture was incubated 24 h at 37 °C, with
shaking at 200 rpm and adjusted to the required concentration. Stocks
of purified ungerminated spores of Ba strain Sterne were prepared as
described previously [38,39]. The spores were incubated in germinant
consisting of 50 mM L-alanine and 100 mM inosine for 25 min at 37 °C,
at which time the spores had uniformly germinated as shown by their
loss of refractility by phase microscopy [38,39]. The spore suspension
was then diluted in buffer or MHB and adjusted to the concentration
required for the assay.
Source References
USAMRIID 33
USAMRIID 36, 37
J. Warawa, NIAID 34
A. Torres, UTMC 32
USAMRIID 35
C.K. Cote, et al.
2.2. Chemicals, antibiotics and peptides
Chemicals were obtained from Sigma-Aldrich (St. Louis, MO) except
as indicated. Antibiotics were procured from Sigma-Aldrich except for
fosfomycin and ceftazidime, which were obtained from USP.
Antimicrobial peptides were acquired from the following sources:
Sigma/Fluka, Bachem (Torrance, CA), Biopeptek (Malvern, PA), and A
&A Labs/Synthetic Biomolecules (San Diego, CA). They included
WLBU2, BMAP-18, mastoparan7, nisin, melittin, magaininII, bacte-
nicin, and CAMA, and their synthesis and activity have been previously
reported [5,7,9,15,20,23]. Stocks of antibiotics and AMPs were pre-
pared in concentrations of 5–10 mg/ml in accordance with manu-
facturer recommendations and stored in single use aliquots at −80 °C.
2.3. Antimicrobial susceptibility tests
2.3.1. Standard assay
In vitro susceptibility assays of antimicrobial MIC were performed in
accordance with Clinical & Laboratory Standards Institute (CLSI) stan-
dards in 96-well microtiter plates with cation-adjusted MHB.
Antimicrobial stock solutions, bacterial inocula, and test conditions
were as described previously [40–43]. Instead of using a vegetative cell
suspension, Ba Sterne inocula were prepared from freshly germinated
spores as specified above. The bacterial inocula were prepared from
suspensions of freshly grown plated colonies, or from an overnight
broth culture (Ft LVS) and adjusted to a final concentration based on the
OD600 reading of 1.5–5 × 105 CFU/ml. Positive growth control wells
included bacteria incubated in buffer without antimicrobial agent. For
assays employing antimicrobial stocks prepared in DMSO, the buffer
used in the control wells contained DMSO at a concentration equal to
that of the highest concentration of DMSO in the antimicrobial-treated
samples to account for any residual antimicrobial activity of DMSO.
MICs were determined by titration dilution covering a broad range of
antimicrobial concentration, typically 0.0312–62.5 μg/ml except as
indicated. After inoculation with the bacteria, the plates were incubated
at 37 °C, except for Yp which was incubated at 28 °C [41]. Yp modifies
its surface LPS structure in various ways, e.g., by reducing the extent of
lipid A acylation, in response to the change in temperature from ≤27 °C
to 35–37 °C. This change may potentially alter the activity or interac-
tion of AMPs with the bacterium, a possibility which was not evaluated
in the current study. However, in the current and previous studies,
outer-membrane active antibiotics and AMPs have been shown to be
active in vitro and in vivo, alone or in antibiotic-peptide combinations,
against gram-negative bacteria to include Yp, as will be described in the
Results and Discussion. In addition, Yp grows faster at 28 °C than at
37 °C, and the three virulence plasmids of Yp are maintained more
stably at 28 °C than at 37 °C [41]. Furthermore, in MIC screens with
numerous Yp strains, Heine et al. [41], observed similar MIC results at
both temperatures; most of the MICs were within one well at the two
temperatures, with only 17.77% (8/45) of the 90% MICs having a two-
well difference [41]. The MIC plates were read 18–24 h post incubation
(except 48 h for Yp and 72 h for Ft) and the MIC determined. The MIC
value was the lowest concentration of antimicrobial agent producing no
visible growth after incubation or the greatest reduction in growth
density, as described below. Minimum bactericidal concentration
(MBC) values were ascertained by plating a 10% volume of the MIC
assay wells lacking visible growth to determine the lowest anti-
microbial concentration producing no growth.
2.3.2. Checkerboard assay
To identify combinations of an antibiotic and AMP which exhibited
synergistic or enhanced antibacterial activity, checkerboard MIC titra-
tion assays were performed by a standard method [44–46]. Briefly,
each antimicrobial was serially titrated two-fold across all the rows or
down the columns in broth microdilution plates; the wells were com-
bined in a third tray which was inoculated with the test organism to
3
Microbial Pathogenesis 142 (2020) 104050
yield the appropriate concentration (1.5–5 × 105/ml) in 100 μl MHB.
The plates were incubated for 24 h at 37 °C (or as indicated in 2.3.1) in
ambient air. One well with no antibiotic was used as a positive growth
control on each plate. Plates were read for visual turbidity to determine
the MIC. The antbiotics tested included nalidixic acid, colistin, cefta-
zidime, tigecycline, minocycline, and novobiocin; and the AMPs tested
included nisin and WLBU2. These data are representative of at least two
separate experiments of similar results per strain. The MIC data were
evaluated for synergistic effects of the combinations by determining
Fractional Inhibitory Concentration Index (FICI) values in accordance
with the following formula: The FICI = FICA + FICB, where
FICA = MIC of drug A in combination/MIC of drug A alone, and
FICB = MIC of drug B in combination/MIC of drug B alone. The FICIs
are then interpreted using these breakpoints for four categories: Sy-
nergy: FICI of <0.5; Additivity: FICI of >0.5 to <1; no interaction
(Indifference): FICI of >1 to <4; and Antagonism: FICI of >4
[45,47–49].
2.4. Direct killing assays
In vitro assays of direct antimicrobial activity were performed in the
absence of bacterial growth according to a modification of the proce-
dure described by Abdelbaqi et al. [18]. Dilutions of selected anti-
microbial agents, alone or combined, were prepared and added in vo-
lumes of 100 μl to wells in triplicate on a 96-well microtiter plate. A
suspension of bacteria was made and adjusted by OD600 in a non-nu-
trient containing buffer to a concentration producing 1.5–5 × 105 CFU
per well (in a final volume of 200 μl). Positive growth control wells
included the bacteria incubated in buffer without antimicrobial agent.
In all assays employing antibiotic/AMP stocks prepared with DMSO, the
latter was added to the control wells as described in 2.3.1. The in-
oculated plates were incubated for 2 h at the appropriate temperature
and serial dilutions then plated in triplicate on solid medium for viable
count determinations. Survival was assessed by comparing the change
in counts of AMP/antibiotic-treated compared to buffer-treated con-
trols. The buffer used for preparing the assay plate and diluting samples
for plating was Gibco PBS (Thermo Fisher Scientific, Waltham, MA,
USA) for all strains except Yp, for which a buffer with 10 mM potassium
phosphate pH 7.4 was prepared and used.
2.5. Time kill assays
Time-associated in vitro killing assays were performed and evaluated
by a modification of previously described methods [43,45,46,50]. In
brief, flasks containing MHB, with or without addition of an anti-
microbial(s), were inoculated with a concentration of bacteria adjusted
as described for the direct killing assays, and incubated with shaking at
200 rpm and at the appropriate temperature. Samples were collected
from the cultures at three to four time points, just prior to incubation
(t0) and after two or three periods of incubation between 6 h and 72 h
depending on the strain. Viable counts were determined by serial di-
lution and plating.
2.6. Statistics
The statistical significance (P < 0.05) of viable counts acquired in
Direct Killing or Time Kill assays were determined by t-tests with the
Holm-Šídák method to correct for multiple comparisons, or by ANOVA
and pairwise multi-comparison post-test (e.g., Tukey's). These statistical
analyses were done with GraphPad Prism versions 7.0 or 8.1.
3. Results
3.1. Antimicrobial susceptibility to antibiotics and AMPs
MIC assays were performed to screen for antibiotics and AMPs
C.K. Cote, et al. Microbial Pathogenesis 142 (2020) 104050

Table 2 Table 2 (continued)

Microbial sensitivity to antibiotics and antimicrobial peptides.
Bacterial strain Antimicrobial agent MICa - mean (range)b MBCa
Bacterial strain Antimicrobial agent MICa - mean (range)b MBCa

Antibiotics Peptides
Bp JW270 ceftazadime 3.91 Ndc Bp JW270 WLBU2 >62.5 (31.25->125) >62.5
E. coli ATCC 25922 0.092 (0.061–0.122) Nd E. coli ATCC 25922 5.60 (1.95–17.7) Ndc
Ba Sterne >31.2d >31.2 Ba Sterne 7.81 (3.91–15.62) Nd
Yp CO92 pgm- pPst- 0.122 0.122 Yp CO92 pgm- pPst- 31.25 31.25
Ft LVS strR 0.0305 Nd Ft LVS strR 9.76 (3.91–15.62) 31.25
Bm CLH001 3.91 (1.95–7.81) Nd Bm CLH001 62.5 (31.25->62.5) Nd

Bp JW270 fosfomycin > 62.5 Nd Bp JW270 BMAP18 >62.5 Nd

E. coli ATCC 25922 15.62 Nd E. coli ATCC 25922 7.81 Nd
Ba Sterne 31.25 Nd Ba Sterne 15.62 Nd
Yp CO92 pgm- pPst- 31.25 Nd Yp CO92 pgm- pPst >62.5 >62.5
Ft LVS strR > 62.5 Nd Ft LVS strR 1.95 Nd
Bm CLH001 > 62.5 Nd Bm CLH001 >62.5 Nd

Bp JW270 colistin > 62.5 Nd Bp JW270 Mastoparan7 >62.5 >62.5

E. coli ATCC 25922 1.95 Nd E. coli ATCC 25922 3.91 Nd
Ba Sterne 62.5 Nd
Yp CO92 pgm- pPst- >62.5 >62.5
Bp JW270 aztreonam 31.25 Nd
Ba Sterne >62.5 Nd
Yp CO92 pgm- pPst- 0.03 Nd Bp JW270 Nisin >625 (625–774) Nd
Ft LVS strR 31.25 Nd E. coli ATCC 25922 >625 (625–774) Nd
Bm CLH001 31.25 Nd

Bp JW270 Melittin >62.5 Nd

Bp JW270 nalidixic acid 7.81 (3.91–15.62) Nd E. coli ATCC 25922 62.5 Nd
E. coli ATCC 25922 1.95 (<0.98–1.95) Nd
Ba Sterne 3.91 Nd
Yp CO92 pgm- pPst- 15.62 Nd Bp JW270 Bactenicin >62.5 Nd
Ft LVS strR 0.487 Nd E. coli ATCC 25922 >62.5 Nd
Bm CLH001 31.25 Nd

Bp JW270 MagaininII >62.5 Nd

Bp JW270 minocycline 2.94 (1.95–3.91) >62.5 E. coli ATCC 25922 >62.5 Nd
E. coli ATCC 25922 0.102 (0.061–0.122) Nd
Ba Sterne 0.041 (0.031–0.98) >62.5
Yp CO92 pgm- pPst- 1.63 (0.98–1.95) >62.5 Bp JW270 CAMA >62.5 Nd
Ft LVS strR 0.061 Nd E. coli ATCC 25922 62.5 Nd
Bm CLH001 1.37 (0.98–1.95) Nd
a
The antimicrobial measures (MIC and MBC) are described in the Materials
and Methods. Values are μg/ml.
Bp JW270 doxycycline 2.93 (1.95–3.91) >31.25 b
Values with no range indicate identical MIC values were obtained in dif-
E. coli ATCC 25922 0.244 Nd
Ba Sterne 0.98 >62.5
ferent experiments.
c
Yp CO92 pgm- pPst- 0.98 >62.5 Nd - Not done.
d
Ft LVS strR 0.122 Nd The signs indicate limits of detection (LOD): ">" indicates the highest
Bm CLH001 0.98 Nd concentration tested, and "<" gives the lowest tested.

Bp JW270 tigecycline 2.93 (1.95–7.82) Nd

having potential use in broad-spectrum therapeutic combinations. Eight
E. coli ATCC 25922 0.244 Nd early generation antibiotics were selected, and MICs for surrogate
Ba Sterne 0.349 (0.12–0.98) >15.62 strains of Ba, Yp, Bp, Bm, and Ft (Table 1) were determined. Two ad-
Yp CO92 pgm- pPst- 0.98 (0.49–1.95) >15.62 ditional antibiotics in wide current use, doxycycline and ceftazidime,
Ft LVS strR 0.244 0.98
were included as controls. Three of the antibiotics had low to moderate
Bm CLH001 15.62 (7.81–31.25) Nd
MICs for the five strains and the Ec standard strain: novobiocin, tige-
cycline and minocycline; the latter two belong to the tetracycline fa-
Bp JW270 novobiocin 1.63 (0.98–1.95) Nd mily. Exceptions included the relative resistances of Bm to tigecycline
Ba Sterne 0.82 (0.49–1.95) Nd
(15.62 μg/ml) and Yp to novobiocin (31.25 μg/ml), as shown in
Yp CO92 pgm- pPst- 31.25 Nd
Ft LVS strR 0.046 Nd Table 2. The Burkholderia, were overall the most resistant of the five
Bm CLH001 1.30 (0.98–1.95) Nd species, as shown in Table 2. Most antibiotics had similar MICs for Bm
and Bp, however Bm was more resistant than Bp to nalidixic acid and
tigecycline, whereas Bp was more resistant to doxycycline and possibly
Bp JW270 fusidic acid 31.25 Nd
Bm CLH001 31.25 Nd
minocycline.
Of the eight peptides tested, only WLBU2 and BMAP-18 showed
activity against the Burkholderia and the other surrogate strains tested
(Table 2). Subsequent work centered on WLBU2, a cationic 24 amino
acid helical peptide which had been shown previously to exhibit broad-
spectrum activity against Ft, Bp, and Yp, and to be effective against
intracellular infection without host cell cytotoxicity [18,20]. It was

4
C.K. Cote, et al.
observed that in the MIC assays, AMPs such as WLBU2 and some bac-
teriostatic antibiotics did not completely eliminate production of visible
growth by the bacteria, especially the Burkholderia spp., but produced
large reductions in growth. The MIC was defined as the concentration
which inhibited visible growth or produced the largest reduction in
growth density by visual inspection and with selected confirmation by
viable count determinations or OD600 readings [40–42].
3.2. Antimicrobial activity of therapeutic combinations
3.2.1. Checkerboard MIC assays
Based on these results, a preliminary set of effective candidates for
evaluation as combination therapeutics was identified, which included
the antibiotics tigecycline, minocycline, or novobiocin and peptide
WLBU2. The extent of synergistic or enhanced antibacterial activity of
these down-selected combinations was characterized by three methods,
checkerboard MIC, direct killing assays, and time kill kinetic assays.
Improved activity could potentially allow reductions in the effective
Table 3
Summary of the effects on antibacterial activity of AMP-antibiotic combinations.
5
Microbial Pathogenesis 142 (2020) 104050
concentrations of the individual constituents thereby lessening their
toxicity. The checkerboard MIC data were analyzed by determining the
FICI values. The FICI results for the three combinations for five species
are available as supplementary tables (Tables A1, A.2, and A.3). Each of
the three antibiotic-AMP combinations was effective (synergistic or ad-
ditive) against at least three strains, as determined by a reduction in MIC.
Considered together, the minocycline- or tigecycline-peptide pairs in-
creased the antibacterial sensitivities of Yp, Ft, Ba and Bp (Table 3). The
novobiocin-WLBU2 combination augmented the susceptibility of all five
strains (Table 3). The Burkholderia were the most resistant strains overall,
especially Bm; i.e., the two tetracycline-peptide combinations failed to
increase Bm sensitivity beyond that of either alone. However, the high
sensitivity of Bm CLH001 to minocycline (and novobiocin) may have
partially prevented any additional antibacterial effects of the peptide. In
addition to the selected antimicrobial combinations, assays were per-
formed to consider other potential antimicrobial candidates. Combina-
tions of WLBU2 and nalidixic acid caused no change in the MIC for Ec but
afforded an 8-fold reduction in MIC for Bp; and the AMP nisin combined
C.K. Cote, et al.
Fig. 1. Direct killing of Ba by peptide WLBU2 in buffer. Germinated spores of
Ba were suspended in PBS, incubated with dilutions of the AMP, and plated for
viable counts. Data are the mean CFU/ml and SD (error bars). All five con-
centrations of WLBU2 reduced Ba viable counts compared to the untreated
control, p < 0.0001. From highest to lowest WLBU2 concentrations, viable
counts ranged from 17.6% to 50.1% of the diluent control. The difference be-
tween mean counts of the highest (31.25 μg/ml WLBU2) and lowest (1.95 μg/
ml) dose groups was significant, p = 0.0017.
with either colistin or nalidixic acid resulted in no change in antibacterial
activity for Ec or Bp (data not shown).
3.2.2. Direct killing tests
Antimicrobial peptides have been shown to directly kill bacteria in
the absence of growth [1,4,5,8,18,20]. To assess direct killing by WLBU2,
fresh cultures of the bacteria were suspended in buffer (2.4), incubated
for 2 h with different peptide concentrations, and dilution plated for
viable counts. The bactericidal activity of the peptide was not always
proportional to the dose and inter-experimental variability was occa-
sionally observed. However, the inclusion of untreated controls in all
experiments allowed the effects of the treatments on viability to be de-
termined within each experiment. As reported previously [18], our
strains of Yp and Ft were sensitive to killing by WLBU2. Treatment of Ft
with 15.62 μg/ml WLBU2 caused a mean of 61% killing compared to the
control. Exposure of Yp to 0.98 μg/ml (0.03x MIC) resulted in a 70%
reduction in viability in these tests compared to the untreated control
(data not shown). Similar to Yp, Ba was also very sensitive to the peptide
6
Microbial Pathogenesis 142 (2020) 104050
(Fig. 1). A WLBU2 dose of 0.25x MIC reduced Ba viability by >50%. The
Burkholderia were again the least sensitive of the five strains tested, as
predicted by the sensitivity results (Table 2). In agreement with previous
findings [18], Bp was partially killed by WLBU2, which caused ap-
proximately 35% killing at concentrations from 7.81 to 62.5 μg/ml. The
extent of direct killing of Bm by WLBU2 was also low, ranging from none
to a 37% reduction at high doses of ≥31.25 μg/ml.
To determine if the addition of an antibiotic could enhance direct
killing, the assays were performed with the peptide in combination with
tigecycline, minocycline, or novobiocin. Antibiotics with bacteriostatic
activity such as the tetracyclines are often only effective against re-
plicating and/or metabolizing bacteria [30,51]. However, tetracycline
can alter cytosolic membranes and directly cause cell leakage [52], and
DNA gyrase inhibitors such as novobiocin can exhibit concentration-de-
pendent bactericidal effects [53]. Furthermore, the effects of WLBU2 on
membrane integrity could potentially increase antibiotic uptake [4,54].
The results of the direct killing assays partially supported the enhanced
antibacterial efficacy of the antibiotic-peptide combinations (combos)
and emphasized the importance of identifying the optimal concentrations
of both components. As summarized in Table 3, the direct killing data did
not always parallel the MIC data (using growing cultures), as expected.
The tetracycline-peptide combos augmented direct killing of Ba, Bp, and
Bm; and the novobiocin-WLBU2 combo enhanced killing by Bp. None of
the combos augmented the direct killing of Yp, which was very sensitive
to WLBU2 alone in the three assays (Table 3). Fig. 2 illustrates the in-
crease in killing of Bp and Bm afforded by combining tigecycline and
WLBU2 in the checkerboard and killing assays. Whereas tigecycline
alone significantly reduced Bp viability (p < 0.0001), WLBU2 did not
(Fig. 2A). The tigecycline-associated killing was further enhanced by
combining 0.98 μg/ml tigecycline (0.33x MIC) with WLBU2
(p = 0.0009) but not by the other four combo treatments. Thus, effects
of the peptide were dependent on concentration but was not directly
dose-related. The results suggested that the activity for Bp of a low
concentration of tigecycline (0.33x MIC) could be significantly enhanced
by the addition of WLBU2. Also, the reduced bactericidal activity of the
combos containing higher concentrations of tigecycline (p = 0.002 for
the 3.91 μg/ml combo) suggested that the peptide might have antag-
onistic effects on tigecycline depending on the concentration of each.
Whereas neither antimicrobial alone significantly impacted Bm viability
(Fig. 2B), the peptide combined with tigecycline from 3.91 to 15.62 μg/
ml significantly increased killing.
3.2.3. Time kill assays
To analyze time-associated antimicrobial killing, flasks with MHB,
alone or with antimicrobials present, were inoculated and cultures were
sampled at three to four time points for viable counts. The findings are
Fig. 2. Direct killing of Burkholderia by tigecycline
and peptide WLBU2. A. Bp. WLBU2 and tigecy-
cline were tested separately (31.25 μg/ml or
0.98 μg/ml, respectively), or in five combinations.
The combos contained WLBU2 (31.25 μg/ml) and
tigecycline ranging from 0.015 μg/ml to 3.91 μg/
ml, as indicated in the X axes. The combo reduced
viability more than did tigecycline alone
(*p = 0.009). B. Bm. The combos contained
WLBU2 (31.25 μg/ml). WLBU2 alone and tigecy-
cline alone (3.91 μg/ml or 15.62 μg/ml) did not
significantly reduce the viable counts compared to
the control. The combos with tigecycline from
3.91 to 15.62 μg/ml significantly reduced viability
compared to the control (**p ≤ 0.002).
C.K. Cote, et al. Microbial Pathogenesis 142 (2020) 104050

Fig. 3. Time course assays of killing of three strains by a tetracycline-peptide combination. The change in viable counts with incubation time in MHB with or without
an antibiotic or WLBU2 are shown. A. Yp pgm-pPst-was treated with minocycline (1.46 μg/ml) and/or WLBU2 (3.91 μg/ml). The combo group counts were less than
the other groups at 24 h and 48 h, with p values from 0.0087 to <0.0001. B. Ft LVS was treated with tigecycline (0.488 μg/ml) and WLBU2 (3.78 μg/ml). The combo
treatment counts were less than those of the other groups for the 24 h and 72 h samples, p values from <0.0001 to 0.033. C. Ba was treated with minocycline
(0.328 μg/ml) and/or WLBU2 (1.96 μg/ml, 0.25x MIC). The counts of the combo-treated group were less or nearly significantly less than those of minocycline alone
at 6 h and 24 h (p < 0.0001) and WLBU2 alone at 6 h (p = 0.063) and 24 h (p < 0.0001).

Fig. 4. Time course assays of killing of Bp by novobiocin-peptide combinations: effects of growth conditions on antibacterial activity. A. Treatment with WLBU2 and
novobiocin (6.52 μg/ml) and incubation in standard conditions. The viable counts of the combo at 24 h were less than those of the novobiocin alone (p = 0.0076)
and the WLBU2 or untreated groups (p = 0.0001). B and C. Treatment with WLBU2 and novobiocin (13.04 μg/ml). B. Incubation was in MHB prepared by standard
procedures. WLBU2 treatment had no effect on killing. C. Incubation was in nutritionally suboptimal MHB. At 24 h and 48 h, the combo counts were reduced
compared to those of the WLBU2 treated (p ≤ 0.009) and of the untreated and novobiocin treated groups (p ≤ 0.0058).

summarized in Table 3 and illustrated in Fig. 3, Fig. 4, and Fig. 5. The treatments were bactericidal for Yp at 24 h, and the combo continued
tetracycline-AMP combos had significantly enhanced antibacterial ac- killing to 48 h (Fig. 3A). WLBU2 alone temporarily retarded growth to
tivity against Ba, Ft, and Yp (Fig. 3). The minocycline alone and combo 24 h. The tigecycline-WLBU2 combination significantly impeded

7
C.K. Cote, et al. Microbial Pathogenesis 142 (2020) 104050

Fig. 5. Time course assays of killing of Bm by antibiotic-peptide combinations. The change in viable counts of Bm with incubation time in FeSO4-supplemented MHB.
A. WLBU2 (31.25 μg/ml) had no effect on, or antagonized killing by, novobiocin (6.53 μg/ml) after 48 h incubation. B. Treatment with tigecycline (15.62 μg/ml) and
WLBU2 (7.81 μg/ml). At 6 h, the combo counts were less than those of the WLBU2-treated and untreated (p = 0.0004) but not the tigecycline-treated (p = 0.241)
samples. At 24 h, the combo counts were less than those of tigecycline (p = 0.0021), WLBU2 (p < 0.0001), and untreated (p = 0.0004) samples. C. The conditions
were the same as B except the iron concentration was decreased two-fold. Tigecycline killing was increased 240–fold compared to that in panel B.

growth of Ft LVS compared to the other three groups at 24 h and 72 h
(Fig. 3B). A similar enhancing effect of a minocycline-WLBU2 combo
was observed with Ba (Fig. 3C). Compared to the other three groups,
the combo treatment caused the greatest reduction in viability at both
the early (6 h) and last timepoint (Fig. 3C). The combo-treated values
remained less than those of the minocycline-treated, though the two-
fold difference was no longer significant by 24 h.
Neither tetracycline-peptide combo positively impacted Bp killing,
and in some circumstances antagonized the antibacterial effects of the
antibiotic (Table 3). However, the novobiocin-WLBU2 combo sig-
nificantly reduced viability of all five strains except Bm (Table 3). As
shown in Fig. 4 (and Table 3), peptide WLBU2 enhanced the killing of
Bp in combination with a lower concentration of novobiocin (Fig. 4A)
or had no effect on killing associated with a larger concentration
(Fig. 4B). The performance of the assay under suboptimal growth
conditions greatly enhanced the killing of Bp by novobiocin-peptide. In
Fig. 4C, Bp was incubated in MHB medium which had been subjected to
a longer than recommended period of steam sterilization. It was nu-
tritionally inadequate as shown by the absence of growth by the un-
treated control after 6 h. However, at 24 h and 48 h, the combo counts
were greatly reduced compared to the other three groups. The growth
conditions described in Fig. 4C could resemble that occurring in in vivo
niches which induce a dormant or persistent bacterial state such as the
host cell phagolysosome. In such circumstances, combination anti-
microbials might be especially effective therapeutics, as elaborated
upon later. Fig. 5 illustrates the effects of two antibiotic-peptide combos
on Bm viability. Bm CLH001 was very sensitive to novobiocin (Fig. 5A).
WLBU2 did not augment killing but instead appeared to antagonize
antibiotic killing at high concentration. In contrast, the peptide en-
hanced the tigecycline-associated decrease in Bm viability (Fig. 5B).
Interestingly, when the iron-deficient Bm CLH001 was incubated in
medium with a suboptimal iron concentration, the peptide did not
enhance tigecycline killing, however, the activity of tigecycline alone
was increased 240–fold (Fig. 5C). These findings again suggested that
antibacterial effects of the therapeutics observed in vitro might differ
from those occurring in the less optimal growth environment.
4. Discussion
In this study, antibiotic-AMP combinations were identified as po-
tentially useful alternative countermeasures for treating infections by
five bacterial biothreat species, including the AMR Burkholderia bac-
teria. The three down-selected combinations consisted of a tetracycline
(tigecycline or minocycline) or gyrase inhibitor novobiocin, each
combined with a cationic AMP (WLBU2). No single antibiotic-AMP
combination exhibited synergistic or enhanced antibacterial activity
against all five bacterial agents in all three assays. However, based on
the results of these antimicrobial sensitivity measures, at least one
specific treatment (and an alternate) were effective for each strain: AMP
with novobiocin (or with minocycline) for Ba and Yp, AMP with no-
vobiocin (or tigecycline alone) for Bp, AMP with novobiocin (or with
tigecycline) for Ft, and AMP with tigecycline for Bm. Novobiocin alone
was also an effective alternate antimicrobial for Bm, but due to toxicity
associated with this antibiotic (discussed below), this is not a realistic
therapeutic option.
The advantages of managing complex infections with a combination
of antibiotics is well established [55]. This strategy is valuable when the
components display synergistic or additive effects in vitro; the microbial
etiology cannot be determined, is polymicrobial, or is caused by an
AMR strain; or the infection is prone to an extended course and emer-
gence of resistance. The efficacy of antibiotic combination therapy is
supported by studies performed in clinical and in vivo settings
[48,49,55–58]. However, the current escalation in bacterial resistance
to conventional antibiotics has stimulated an urgent search for alternate
therapeutics.
Antimicrobial peptides are good candidates to consider in an al-
ternate antibiotic-AMP dual treatment approach. Hallmarks of AMPs
include their broad and rapid activity against a diverse range of

8
C.K. Cote, et al.
pathogens. The mechanisms, which can be both direct and indirect,
underlying the potent antimicrobial action of AMPs have been widely
studied [1,2,59]. The major mode of direct bacterial cytotoxicity occurs
by perturbation of the bacterial outer membrane. Natural AMPs com-
monly have an amphipathic conformation with positively charged and
hydrophobic groups present on opposing faces of the structure, which
imparts the ability to electrostatically disrupt a phospholipid membrane
as found on bacterial surfaces [1,2,10]. The mechanisms for this are not
well understood but models describe the creation of membrane pores
with subsequent insertion of the AMP and leakage of ions and meta-
bolites and ultimately the loss of membrane function; alternately,
membrane absorption of the AMPs as a layer, followed by membrane
solubilization and disintegration, has been described [1–3,59]. Some
AMPs penetrate cells by other means which involve bacterial cell pe-
netration as a result of the affinity of AMPs for LPS or the facilitation by
proline moieties of phospholipid interactions by AMPs [2,60]. Upon cell
entry, the AMPS interfere with critical intracellular functions including
RNA and protein synthesis and other processes [2,61]. These various
anti-bacterial activities of AMPs are not mutually exclusive, a property
which likely affords AMPs the ability to evade bacterial resistance
mechanisms [62].
In addition to the direct bacterial killing, many AMPs exhibit in-
direct function through immunomodulatory activities, i.e., the stimu-
lation of chemotaxis, differentiation of immune cells, and regulation of
inflammatory cell responses and cell death pathways. Related host-
protective functions include the ability to neutralize LPS and disrupt
biofilms [1,2,59,63]. Both the direct and indirect AMP actions can
contribute to protective host responses to invading pathogens. Thus,
AMPs are often referred to as host defense peptides (HDP).
In contrast to conventional antibiotics, AMPs have a number of
properties that confer the ability to overcome common mechanisms of
multi-antibiotic resistant pathogens. Many AMPs do not require meta-
bolically active, replicating organisms to kill; and they inactivate bac-
teria rapidly which limits time for mutants to predominate. Also, some
AMPs act on more than one host target, as mentioned above
[2,54,59,64–66]. However, bacteria can develop resistance to AMPs,
though this is expected to be much less common than resistance to
antibiotics and more difficult for bacteria to acquire [2,3,54,59]. As an
example of an AMP resistance mechanism, changes in bacterial outer
membrane lipoteichoic acid can occur which reduce its negative charge
and thus binding by AMPs [3].
Although AMPs have long been attractive as potential anti-
microbials due to their aforementioned properties, there are few reports
demonstrating actual therapeutic in vivo efficacy of AMPs. Protection of
mice was afforded by defensins against lethal infection with P. aerugi-
nosa; infection by Ba spores or exposure to anthrax lethal toxin; and
infection by carbapenem-resistant clinical isolates of Acinetobacter
baumanni [4,19,64,67,68]. Although various AMPs are in human clin-
ical trials, only a few FDA-approved products are available, though
rarely used (e.g., the polymixins). This paucity of efficacy data is par-
tially due to toxicity concerns and also to other known and potential
drawbacks of the natural AMPs. For instance, some AMPs lack specifi-
city, e.g., bind unintended host targets in tissues and serum, leading to
toxic effects as manifested, i.e., by lysis of erythrocytes due to mem-
brane permeation and disruption [16,54,87]. Furthermore, their anti-
microbial activity can be inhibited in the presence of acidic pH, sodium
ions, divalent cations, proteases, and other substances, thus reducing
the level of free peptide available for antimicrobial use [16,17,69,87].
These limitations can be surmounted to a large extent by structural
optimization. AMP modifications can be made which theoretically
cause them to be antibacterial at concentrations well below those which
are lytic for host cells and also to more narrowly target bacteria
[9,14,19,20,62,63,70,87,88]. Several approaches have also been uti-
lized to improve the cell selectivity of AMPs. These include the opti-
mization of physicochemical properties of peptides, constraint of AMP
conformations, the use of unusual amino acids or of D-stereoisomer or
9
Microbial Pathogenesis 142 (2020) 104050
substituted (e.g., fluorinated) amino acids, and the modification of
peptides by polymers [9,14,19,20,62,63,70,87,88]. Also, AMPs can be
rendered less harmful to the host by minimizing their interaction with
host tissues. Epithelial cells are more resistant to AMP toxicity and in-
jection of the peptides intracutaneously might obviate some of their
toxicity [87]. Similarly, encapsulation and delivery of AMPs to pha-
gocytic leukocytes could conceivably allow a peptide to primarily bind
to and kill phagocytosed bacteria. A peptide optimization approach is
discussed further below for WLBU2.
Finally, since AMPs are often insoluble in water and must be dis-
solved in a solvent such as DMSO, it is important to consider the anti-
host effects of residual levels of solvent in the antimicrobial inocula. In
discerning the most effective combinations of AMP and antibiotic in our
study, we selected those with total final DMSO concentrations below
those which inhibited the bacteria, as indicated above. The toxicity of
DMSO for humans and animals is also a concern but is not well defined.
It is considered to be slightly hazardous for humans in case of inhala-
tion and contact of full strength DMSO with the skin and eyes [89,90].
It has been approved for use by the FDA for specific clinical conditions
such as inflammatory genitourinary disorders and has been used to
treat numerous other conditions [90,91]. In mice DMSO has a max-
imum tolerated intravenous dose (MTD) of 40% (2200 mg/kg) and a
maximum to-observed-effect dose (NOEL) of 30% (1650 mg/kg)
[92,93]. In our combination assays, DMSO was used to prepare con-
centrated stocks of WLBU2, and residual levels of the solvent were
present in final amounts ranging from 0.04% to 0.625%, as discussed
further below.
The combination of AMPs with an antibiotic may serve to reduce the
toxicity and off-target activity of the peptide. Naghmouchi and cow-
orkers showed that combinations of the peptides nisin A or pediocin
with colistin were synergistic, thus permitting a reduction in the ef-
fective concentrations (and thus toxicities) of each component [23];
this finding was confirmed and extended in recent studies employing
other AMPs and standard antibiotic drugs of choice [54].
Although synergistic combinations of an antibiotic and AMP pro-
vide a promising approach for combatting infections, the basis of this
synergy is not well understood. A discussion of the many established
and proposed mechanisms is beyond the scope of this paper and are
reviewed in detail elsewhere [54]. The major way by which AMPs with
strong membrane permeabilizing activity enhance susceptibility is to
increase the access of antibiotics to their host cell targets; yet as noted
above, some AMPs can act solely or primarily on internal bacterial
targets. Furthermore, the synergy observed for antibiotic-AMPs is not
necessarily unidirectional. In addition to the positive effects of AMPs on
antibiotic activity, the antibiotic may cause alterations in the host cell
which, for instance, improve the incorporation or orientation of AMPs
in the bacterial membrane [54].
In the current study, we distinguished licensed but rarely used an-
tibiotics which in combination with an AMP enhanced the susceptibility
of five Tier 1 select agents. Three candidates were identified as proto-
types of effective broad spectrum therapeutics, and consisted of the
peptide WLBU2 and the antibiotics tigecycline, minocycline, or novo-
biocin. The eight AMPs screened initially were chosen based on the
literature, as described in the Introduction and previous studies
[1,5,7,18,70], with specific emphasis on Burkholderia spp. due to their
high-level of intrinsic antibiotic resistance. In killing assays with ten
AMPs, Kanthawong detected low to moderate sensitivity to killing of Bp
but at high concentrations only (100 μM–200 μM). The exception was
LL37, which caused a large, rapid decrease in viability of Bp exposed to
200 μM peptide. This LL37 sensitivity was not observed by other
workers and is probably Bp strain-related [5,7,18].
The engineered peptide WLBU2 was down-selected as the AMP
component of a combination therapeutic based on the MIC and
checkerboard assay findings. The engineering of novel peptides from
native progenitors to maximize potency and specificity for anti-
microbial targets is an established tactic [9,14,19,20,70]. Unlike six of
C.K. Cote, et al.
the seven other AMPs, WLBU2 displayed antibacterial activity against
all five agents (Table 2; and data described in the text). The suscept-
ibilities of the five biothreat species were greater for WLBU2 than for
BMAP-18, except for Ft LVS. Its selection is also supported by previous
work to construct and characterize WLBU2. This 24-aa cationic peptide,
composed of valine, arginine, and tryptophan residues, was one of
several derivatives of a lytic peptide base. WLBU2 had the highest ac-
tivity against Pa. It possessed broad spectrum activity against Yp, Ft,
and Bp and killed intracellular Ft in macrophage assays [18]. The
peptide retained its activity in serum and blood; killed Pa in co-cultures
with human skin fibroblast without cell toxicity; prevented biofilms on
primary human airway epithelial cells; and protected mice against in-
fection when administered by intravenous or aerosol routes
[4,19,20,63]. The structure of and the rate of killing by WLBU2 sup-
ported the conclusion that it activity was due primarily to direct
membrane disruption, as occurs for the human AMP LL37 [1,71].
However it is possible that the peptide has ancillary intracellular effects
on bacterial processes (synthesis, transcription or translation), as de-
scribed for some defensins and cathelicidins [61,72] or it has other
immunomodulatory effects on host responses. Studies have docu-
mented both the loss and retention of WLBU2 activity in the presence of
RBCs [16,19]. The importance of this or other potential limitations to
its clinical use may require further tests of in vivo efficacy in animal
models.
Based on previous in vitro and in vivo data, there is ample evidence
to support the premise that clinical efficacy of the peptide-antibiotic
combinations against biothreat pathogens is possible at achievable yet
nontoxic doses. As described above, Deslouches and coworkers
[4,18–20,59,63,94,95] have evaluated the effects of WLBU2 on multi-
drug resistant “ESKAPE” pathogens such as Pa, a common pulmonary
and systemic pathogen which is often refractory to treatment. In later
studies, as little as 16–20 μM WLBU2 prevented Pa biofilm formation on
primary human airway epithelial cells; and concentrations up to at least
50 μM were not toxic for the cells as shown by no change in transe-
pithelial electrical resistance [4,63]. Furthermore, WLBU2 (20 μM) also
significantly enhanced killing of Pa in this biofilm model by sub-
inhibitory concentrations of antibiotics which are currently used in
clinical treatment of biothreat agents (ciprofloxacin, meropenem, cef-
tazidime). Finally, Chen et al. achieved protection of mice against
pneumonia by WLBU2 delivered intratracheally after infection with Pa
[4]. The lowest therapeutic dose was determined to be equivalent to
0.05 mg/kg (1.25 μg/25 gm mouse), which was 280-fold below the
maximum tolerated (nontoxic) dose of peptide, determined to be
14 mg/kg [94] in mice given WLBU2 intraperitoneally or intravenously
after exposure to a lethal dose of Pa. WLBU2 reduced both the bacterial
load (by 2 logs) and the extent of its induced inflammation with no
signs of disease or AMP-associated toxicity. Recently, Abdelbaqi and
coworkers demonstrated efficacy of the peptide against Bp, Ft, and Yp;
and the killing of Ft within infected macrophages with WLBU2 was
achieved by 12.5 μM–25 μM [18]. In our assays, antibiotic activity
against the five bacterial agents was significantly enhanced by WLBU2
at 2.0–31.25 μg/ml (approximately 0.5–10 μM), which is well within
the range of effective concentrations of this peptide as described above.
The three antibiotics selected for evaluation as the antibiotic com-
ponent of combination therapeutics were chosen based on their prop-
erties and their activity in this study. They are older licensed antibiotics
which are not in high use currently, but have the potential for inclusion
in combination therapies. The results of MIC screening, checkerboard
assays, and functional tests supported the latter premise.
Tigecycline and minocycline are members of the tetracycline family,
broad spectrum polyketide antibiotics produced by Streptomyces which
are bacteriostatic protein synthesis inhibitors. They inhibit the initia-
tion of translation in various ways by attaching reversibly to the 30S
ribosomal subunit (composed of 16S rRNA and 21 proteins) and then
inhibiting the binding of aminoacyl-tRNA to the mRNA translation
complex [43,49,53,55]. Tigecycline is a glycylcycline derivative which
10
Microbial Pathogenesis 142 (2020) 104050
has an extended range against gram-positive and –negative bacteria
[73]. Due to its large additional side chain, it can avoid some of the
common tetracycline resistance mechanisms and is active against many
tetracycline-resistant strains. Specifically it can overcome resistance
caused by at least one of the tetracycline efflux pumps and by the
bacterial ribosome protection protein, but not due to the tetracycline
inactivation enzyme or the RND antibiotic efflux pump [73,74]. It has
improved the outcome of infection with carbapenem-resistant and XDR
Acinetobacter and Klebsiella, when used together with a second anti-
biotic [56–58]). The potential disadvantages of tigecycline include its
bacteriostatic activity in vitro, the possibility of bacterial evolution to
resistance [74], and reported adverse events and clinical failures with
treatment [75]. In the current study, the five strains were all very
sensitive to tigecycline except for Bm (Tables 2 and 3). The strain of Bm
used, Bm CLH001 ΔtonB, Δhcp1, is becoming widely used as a surrogate,
however wild type Bm strains are reported to be sensitive to tigecycline,
e.g., a panel of 23 human and equine clinical isolates of Bm had MICs
ranging from 0.01 to 0.25 μg/ml (unpublished data, USAMRIID Ther-
apeutics core). It is conceivable that an alteration occurred in the outer
membrane of the mutant strain which impeded uptake of the antibiotic.
Therefore, tigecycline may be even more broadly efficacious as part of a
combination therapeutic against wild type Bm than described here.
Minocycline is also broadly effective, has a good pharmacokinetic
profile and is typically safe and well-tolerated, although possibly less so
than doxycycline [76]. However, it was among the most effective an-
tibiotics against three groups which are often difficult to treat, multiply
AMR strains of the Acinetobacter spp complex, the Burkholderia cepacia
complex, and Stenotrophomonas maltophilia [77]. Also, minocycline has
intriguing non-antibiotic biological effects (anti-inflammatory, im-
munomodulatory, and neuroprotective) [76,78], a property which may
augment its value in treating infected patients who are immuno-com-
promised [78]. In the current study, all five species were highly sus-
ceptible to minocycline with MICs ranging from 0.06 to 2.94 μg/ml.
Novobiocin is a member of the aminocoumarin class of antibiotics
[79,80]. It is a bacterial DNA gyrase inhibitor and acts by inhibiting the
GyrB subunit of the enzyme. Novobiocin is bacteriostatic and may be
bactericidal at higher concentrations; however it is rarely used clini-
cally, due to reasons of safety and efficacy, and diminishing commercial
antibiotic development efforts [79]. The potency of novobiocin is
considerably higher than that of the fluoroquinolones which also target
DNA gyrase, but at a different site on the enzyme. Novobiocin is active
mostly against gram-positive bacteria and is an effective anti-staphy-
lococcal agent used to treat MRSA, but it has reduced activity against
many gram-negatives due to their LPS-associated membrane perme-
ability barrier or to specific resistance mechanisms [31,79]. Never-
theless, we observed low novobiocin MICs for not only Ba but also for
Bp, Bm, and Ft (Table 2), in agreement with previous reports [42,81]. In
contrast, Yp was novobiocin-resistant, as described by Heine et al. [41].
Interestingly, in a screen with a small molecule library, Taylor and
coworkers found compounds, termed “antibiotic adjuvants”, which
when combined with novobiocin, appeared to permeabilize the bac-
terial membrane, facilitating novobiocin entry and lowering the MIC
[30]. Similarly, Savage [31] described the ability of cationic outer
membrane permeabilizers to sensitize gram-negative bacteria to hy-
drophobic antibiotics such as novobiocin and thus extend the anti-
bacterial spectrum of these antibiotics [30,31,79]. The enhanced anti-
microbial activity of novobiocin-AMP combinations against the
pathogens, to include Yp resembles that of these antibiotic-enhancing
substances.
Infections which are complicated and difficult to treat with con-
ventional antibiotics include those caused by bacteria which can sur-
vive within host cells or other in vivo niche in a non-growing but viable
state [51]. During certain stages of infection, the five bacterial agents
exist in host cells such as macrophages, and in other in vivo niches such
as biofilms, in a non-replicating but persistent state [82–86]. Bacteria in
these settings are often antibiotic resistant. AMPs which have direct
C.K. Cote, et al.
killing activity may be useful therapeutic components for viable but
dormant bacteria, and their lethal effects might be enhanced by con-
comitant treatment with some antibiotics as suggested (Table 3 and
Fig. 4C). This scenario is supported by pharmacodynamic character-
istics of antibiotics which can cause prolonged antibacterial effects in
vivo on slow- or non-growing bacteria. When serum levels of a bacter-
iostatic antibiotic fall below the MIC, antibacterial effects may linger
due to: persistent suppression of bacterial growth by the therapeutic,
the enhanced susceptibility of treated bacteria to phagocyte killing,
and/or the ability of sub-MIC concentrations of antibiotics to alter
bacterial morphology, slow the bacterial growth rate and extend anti-
biotic suppression [43].
In conclusion, we identified candidate multi-spectrum therapeutics
consisting of specific combinations of an underutilized antibiotic and
antimicrobial peptide which were active against a diverse set of bio-
threat agents. Subsequent efforts will utilize fully virulent strains of the
agents to investigate protective efficacy, host cytotoxicity, and in vivo
availability of the candidates in cell culture and animal models
[18,19,63,87,88]. Finally, it is conceivable that a triple antimicrobial
cocktail of the AMP, novobiocin, and either tigecycline or minocycline
might increase synergy due to the actions of a protein synthesis in-
hibitor and a DNA gyrase inhibitor in combination with the AMP.
CRediT authorship contribution statement
Christopher K. Cote: Investigation, Writing - review & editing.
Irma I. Blanco: Investigation. Melissa Hunter: Investigation. Jennifer
L. Shoe: Investigation. Christopher P. Klimko: Investigation. Rekha
G. Panchal: Supervision, Methodology, Funding acquisition. Susan L.
Welkos: Investigation, Conceptualization, Methodology, Data curation,
Writing - review & editing, Writing - original draft, Visualization,
Supervision, Funding acquisition, Formal analysis, Project administra-
tion, Resources.
Declaration of competing interest
The authors have no conflicts of interest to disclose.
Acknowledgments
The authors thank Drs. J. Bozue and S. Biryukov for their critical
reading of this manuscript.
Funding was provided by JSTO-CBD/DTRA Project CB10647-
Bacterial Biothreat Therapeutics. Opinions, interpretations, conclu-
sions, and recommendations are those of the authors and are not ne-
cessarily endorsed by the U.S. Army.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.micpath.2020.104050.
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