Research Article
www.acsami.org
Chitosan Microparticles Exert Broad-Spectrum Antimicrobial Activity
against Antibiotic-Resistant Micro-organisms without Increasing
Resistance
Zhengxin Ma,†,‡ Donghyeon Kim,‡,§ Adegbola T. Adesogan,‡ Sanghoon Ko,∥ Klibs Galvao,⊥,#
and Kwangcheol Casey Jeong*,†,‡
†
Emerging Pathogens Institute, University of Florida, Gainesville, Florida 32611, United States
Department of Animal Sciences, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611, United
States
§
Division of Applied Life Science (BK21plus, Insti. of Agri. & Life Sci.), Gyeongsang National University, Jinju, South Korea
∥
Department of Food Science and Technology, Sejong University, Seoul, South Korea
⊥
Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610,
United States
#
D. H. Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, Florida 32610, United States
‡
ABSTRACT: Antibiotic resistance is growing exponentially,
increasing public health concerns for humans and animals. In
the current study, we investigated the antimicrobial features of
chitosan microparticles (CM), engineered from chitosan by
ion gelation, seeking potential application for treating
infectious disease caused by multidrug resistant microorganisms. CM showed excellent antimicrobial activity against a
wide range of microorganisms, including clinically important
antibiotic-resistant pathogens without raising resistant mutants
in serial passage assays over a period of 15 days, which is a
significantly long passage compared to tested antibiotics used
in human and veterinary medicine. In addition, CM treatment
did not cause cross-resistance, which is frequently observed with other antibiotics and triggers multidrug resistance. Furthermore,
CM activity was examined in simulated gastrointestinal fluids that CM encounter when orally administered. Antimicrobial activity
of CM was exceptionally strong to eliminate pathogens completely. CM at a concentration of 0.1 μg/mL killed E. coli O157:H7
(5 × 108 CFU/mL) completely in synthetic gastric fluid within 20 min. Risk assessment of CM, in an in vitro animal model,
revealed that CM did not disrupt the digestibility, pH or total volatile fatty acid production, indicating that CM likely do not
affect the functionality of the rumen. Given all the advantages, CM can serve as a great candidate to treat infectious disease,
especially those caused by antibiotic-resistant pathogens without adverse side effects.
KEYWORDS: chitosan microparticles, antimicrobial activity, mutagenesis, multidrug resistance, toxicity
■
INTRODUCTION
The emergence of antibiotic-resistant bacteria has resulted in an
increase in treatment failure rates for infectious diseases leading
to a global public health crisis. The antimicrobial resistance of
bacteria obtained by either mutation or interbacterial
communication causes a high minimal inhibitory concentration
(MIC) of antibiotics, resulting in a decreased susceptible range
for treatment and adverse outcomes.1 The recent phenomenon
of exponential spreading of antibiotic-resistant organisms such
as extended-spectrum β-lactamases (ESBLs),2 Klebsiella pneumoniae Carbapenemase (KPC),3 and methicillin-resistant
Staphylococcus aureus (MRSA)4 threatens public health. Therefore, there is an urgent need to develop alternative therapies to
treat bacterial infections, particularly those caused by multidrug-resistant microorganisms.
© 2016 American Chemical Society
Developing new antibiotics in the 21st century slowed
considerably after over-screening of cultivable soil microorganisms.5 In addition, advanced antibiotic discovery programs including genomics, high-tech chemical approaches, and
high-throughput screening methods have not been successful to
develop new antibiotics and many companies have halted their
antibiotic research programs.6 Nano- and micromaterials have
provided potential for treatment of diseases caused by
antimicrobial resistant microorganisms (ARM). Some metallic
nanoparticles (NP), such as Ag, ZnO, TiO2, Au, Cu, and Al
NPs, kill bacteria using different mechanisms that damage
Received: January 22, 2016
Accepted: April 8, 2016
Published: April 8, 2016
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cellular components, cell wall, and membrane or inhibit enzyme
activity and DNA synthesis.7 However, although metallic NPs
can provide significant benefits in treating infectious disease,
potential toxicity of metallic NPs have limited for clinical use.8
Chitosan, derived by partial deacetylation of chitin, a linear
polymer of β-(1,4)-linked N-acetylglucosamine, is the second
most common polysaccharide found in nature.9,10 It has been
used to make nano- and microparticles for an agent of drug and
vaccine delivery due to its low toxicity, high biocompatibility,
and high loading capacity for hydrophilic molecules including
antibodies due to polycationic properties.11 Chitosan exerts
antimicrobial activity against bacteria and fungi. Although the
mechanisms of the antimicrobial activity are not clearly
understood, it is widely accepted that the bacterial membrane
permeability is altered by interaction with positively charged
chitosan and negatively charged bacterial surface molecules,
resulting in intracellular component leakage that leads to cell
death.12,13 Chitosan has strong antimicrobial activity at acidic
pH, but it is abolished at neutral pH.13 However, chitosan
microparticles (CM), derived from chitosan by ionic crosslinking, showed antimicrobial activity against various pathogens
with different efficacy in the appropriate media not only at
acidic pH but also at neutral pH, where chitosan lose
antimicrobial activity.14 In addition, Jeong et al.15 reported
that CM decreased the shedding of Escherichia coli O157:H7 in
cattle by oral administration. Furthermore, it was found that
CM likely bind to the outer membrane protein OmpA via
hydrogen bonding and LPS via ionic interaction to kill
bacteria.14 However, the mode of action, antimicrobial
property, and potential use of CM against pathogens, especially
antibiotic-resistant microorganisms have not been understood
sufficiently both in vitro and in vivo.
In this study, we further evaluated CM for the antimicrobial
activity and effect of mutagenesis in microorganisms to seek
potential applications in the real world situation. In addition, we
conducted risk assessment of CM treatment in the rumen
microflora to determine if CM can be used for bacterial
infection treatment without causing adverse side effects. It was
demonstrated that CM exert a broad-spectrum bactericidal
activity, particularly against antibiotic-resistant pathogens,
without inducing detectable resistance, suggesting CM could
be a good candidate to treat bacterial infections.
Figure 1. Chitosan microparticles (CM) kill antibiotic-resistant
microorganisms. Live/dead viability assay with 6 different bacteria.
Fluorescent micrograph of cells treated with 0% CM (left), 70%
ethanol (middle), or CM at MIC level (right). ESBL E. coli: extended
spectrum β-lactamase-producing E. coli; KPC: carbapenemasesproducing K. pneumoniae; MRSA: methicillin-resistant S. aureus;
VanR Enterococcus: vancomycin-resistant Enterococcus. The white bar
indicates 10 μm. Results shown are representative of three
independent experiments.
microorganisms tested, including Shiga toxin producing E. coli
O157:H7, ESBL producing E. coli, Carbapenemase producing
K. pneumonia (KPC), methicillin-resistant S. aureus (MRSA),
vancomycin-resistant Enterococcus, and cholera toxin producing
V. cholera. All pathogens treated with CM at MIC level (Table
1) were killed within 2 h incubation. Bactericidal activity
presented as minimum bactericidal concentration (MBC),
defined as eliminating 99.9% bacteria against tested species, was
below 0.004% (40 μg/mL, Table 1). It should also be noted
that CM had excellent bactericidal activity against both Gramnegative and Gram-positive pathogens (Figure 1). Taken
together, these data demonstrated the broad spectrum
antimicrobial activity of CM, especially against antimicrobial
resistant pathogens, providing potential insight as an alternative
treatment method to traditional antibiotics.
CM Do Not Acquire Resistance during Serial Passage.
Acquiring resistance is a key indicator to predict the life span of
newly developed antibiotics, and mutation rate is correlated
with the occurrence of resistance. Consequently, antibiotics
with lower mutation rates may remain effective longer than
those with higher rates. We examined mutation rates in E. coli
O157:H7 following treatment with low levels of CM and the
antibiotic ampicillin. Treated cultures were plated on rifampicin
plates, and colonies growing on the plates were counted to
determine the mutation rates using the MSS maximum
■
RESULTS
Chitosan Microparticles Kill Antibiotic-Resistant Microorganisms. Antibiotic resistance is a challenge for
treatment of infectious diseases and raises rates of mortality
in affected patients significantly.1 It is difficult to develop new
antibiotics to treat infections caused by multidrug resistant
microorganisms.4 We engineered chitosan microparticles (CM)
and the diameter of the prepared CM was about 600 nm with
spherical shape, analyzed by scanning electron microscopy as
described previously.14 To test if CM kill antibiotic-resistant
microorganisms, we conducted the live/dead viability assay
with six clinically relevant pathogens, using microparticles
engineered as described above, including difficult-to-treat
antimicrobial resistant microorganisms (ARMs). As shown in
Figure 1, live bacteria without treatment emit green
fluorescence and are stained with SYTO 9, while dead bacteria
treated with 70% ethanol treatment emit red fluorescence and
are stained with propidium iodide as this dye penetrates into
the cytosol through the damaged membranes. CM had
excellent bactericidal activity against all clinically challenging
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Table 1. Antimicrobial Activity of CM and Antibiotics against Pathogenic Microorganisms
MIC (μg/mL) in MHBa
strains
CM
tetracycline
ampicillin
polymyxin B
MBC of CM in IW (μg/mL)b
E. coli O157:H7
S. uberis
S. enterica
ESBL E. coli
K. pneumoniae
methicillin-resistant S. aureus
vancomycin-resistant Enterococcus
V. cholerae O1 El Tor
V. cholerae non-O1
V. cholerae O395
2000
2000
8000
2000
4000
1000
2000
1000
1000
1000
1
1
Rc
1
2
0.5
1
0.5
0.5
0.5
16
8
16
Rd
R
R
16
32
32
16
2
2
4
4
Re
R
4
R
R
4
10
0.1
0.1
40
1
0.2
0.2
0.4
0.2
0.2
MHB: Mueller Hinton Broth. bIW: isotonic water. cResistant to tetracycline (MIC > 10 μg/mL). dResistant to ampicillin (MIC > 100 μg/mL).
Resistant to polymyxin B (MIC > 10 μg/mL).
a
e
Figure 2. Mutation rate is not increased by CM. (A) Mutagenesis rate of no drug (negative control), 4 μg/mL ampicillin (0.25× MIC, L), 8 μg/mL
ampicillin (0.5× MIC, H), 0.05% CM (0.25× MIC, L) and 0.1% CM (0.5× MIC, H) treatments. Data are means ± SEM of three independent
experiments. Means with different letters differ (P < 0.05). (B) Resistance acquisition during serial passaging in the presence of sub-MIC levels of
CM (solid circle), polymyxin B (open circle), tetracycline (triangle), and ampicillin (square). The highest MIC on each day was plotted. The figures
are representative of three independent experiments.
Figure 3. Sub-MIC levels of CM do not lead to multidrug resistance. Fold change in MIC for kanamycin (cross), ampicillin (square) and tetracycline
(triangle) relative to the no drug treatment in 5 d of culture with 4 μg/mL ampicillin (A) or with 0.05% CM (B). The figures are representative of 3
independent experiments.
likelihood method.16 The mutation rate for untreated E. coli
O157:H7 was approximately 1 × 108/cells per generation
(Figure 2A). Bacteria treated with ampicillin at 0.25× or 0.5×
MIC resulted in significantly increased mutation rates
compared to untreated cells. However, CM treatment at
sublethal levels (0.25× and 0.5× MIC) showed no changes in
comparison to the control, suggesting that the mutation rate
caused by CM is not sufficient enough to promote the
evolution of antibiotic resistance.
To examine if resistance against CM increases during
treatment, we conducted serial passaging of bacteria in the
presence of CM at sub-MIC levels (0.5× MIC). Changes in
MIC in the presence of CM were compared to that of those
with no antibiotic treatment (negative control) and ampicillin
treatment (positive control, Figure 2A). The mutation rate of
CM treatment did not differ from the nontreated control, while
ampicillin treatment increased the mutation rate significantly.
We further tested if the presence of sub-MIC levels of CM over
a long period of time could cause resistance to CM in bacteria
by serial-passage assay (Figure 2B). We could not detect any
resistant mutants during the period of 15 days of passage. In
contrast with CM, sublethal levels of ampicillin, tetracycline and
polymyxin B raised resistance in bacteria within 4 days of serial
passages, indicating CM have an excellent antimicrobial activity
without detectable resistance over long periods of time.
Antibiotic treatment can result in multidrug resistance caused
in part by mutations in drug efflux pumps such as AcrAB.17,18 It
has been shown that bactericidal antibiotics such as βlactamases and quinolones can induce bacteria to generate
reactive oxygen species (ROS). Generation of ROS results in
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Figure 4. Antimicrobial activity of CM in isotonic water. E. coli O157:H7 were grown to early-log (A), late-log (B) and stationary (C) phase, and
about 105 CFU/mL bacteria were inoculated into isotonic water containing different concentrations of CM. E. coli O157:H7 were grown to early-log
(D), late-log (E) and stationary (F) phase, and 5 × 108 CFU/mL bacteria were inoculated into isotonic water containing different concentrations of
CM. Data are means ± SEM of three independent experiments.
stress-induced mutagenesis through DNA damage that activates
the SOS pathway, and then mutation rate is increased when
damaged DNA is repaired by error-prone DNA polymerase.19
This mechanism is, in part, responsible for increased crossresistance against other antibiotics after treatment.19 We tested
if CM would raise cross-resistance against other antibiotics by
measuring the fold increase of MIC after treatment with CM.
When E. coli O157:H7 was treated with ampicillin at 0.25×
MIC (4 μg/mL) for 5 d, the MIC levels of three measured
antibiotics, including kanamycin, ampicillin and tetracycline,
increased up to 2-fold (Figure 3A), whereas bacteria treated
with 0.25× MIC (0.05%) CM did not increase the MIC levels
of all three antibiotics (Figure 3B), indicating that CM do not
raise cross-resistance against other antibiotics.
Antimicrobial Activity of CM. As shown in Figure 1, we
revealed that CM disrupt the bacterial membrane of antibioticresistant microorganisms. It has been shown that CM interact
with outer membrane protein A (OmpA) and lipopolysaccharide (LPS) that results in bactericidal antimicrobial activity.14
Given these notions, we hypothesized that the antimicrobial
activity of CM is not related to bacterial cell wall synthesis, but
directly disrupts bacterial cell walls. To test this hypothesis, we
employed bacterial cells at different growth phases for
bactericidal activity. Log phase cells actively synthesize bacterial
cell walls during multiplication, whereas cell wall syntheses and
cell divisions do not occur in stationary phase cells. Therefore,
CM would not kill stationary phase cells if CM inhibit cell wall
synthesis similar to β-lactam antibiotics. Therefore, we
evaluated antimicrobial activity of CM with bacteria at different
growth phases, early-log, late-log, or stationary phase.
Regardless of bacterial growth phase, CM exerted strong
antimicrobial activity against E. coli O157:H7 (Figure 4).
Bacterial cells at early-log (Figure 4A), late-log (Figure 4B), and
stationary phase (Figure 4C) were effectively killed by CM.
When the early and late-log phase cells were incubated with
CM at the various concentrations, all inoculants (5 × 105 CFU/
mL) were killed within 4 h at 0.001% CM without regrowth at
the later time of incubation. A 5-log reduction was observed
within 6 h of incubation at 0.001% of CM without regrowth of
bacteria until 24 h in the stationary phase cells. Although the
antimicrobial activity was decreased in the stationary phase
cells, CM still exerted a great antimicrobial activity against E.
coli O157:H7 (Figure 4C), resulting in a 4-log reduction. These
data indicate that CM directly disrupt bacterial cell walls that is
likely independent of cell wall synthesis.
When foodborne bacterial pathogens enter host cells, the
numbers of bacteria are generally low (<106 CFU/g of source),
but pathogens will replicate within their niche to reach high
numbers to manifest infectious diseases. To evaluate if CM also
can be applied to treat diseases at the late infectious cycles, we
tested with high numbers of bacteria, 5 × 108 CFU/mL,
mimicking the late stage of infection. The antimicrobial activity
of CM was reproduced with the higher numbers of bacteria
(Figure 4D−F), similar to that with the low numbers of
bacteria (Figure 4A−C). When 5 × 108 CFU/mL bacterial cells
were incubated with CM at 0.004%, a 7-log reduction was
observed within 12 h of treatment. Susceptibility against
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Figure 5. Antimicrobial activity of CM in LB broth. E. coli O157:H7 were grown to (A) early-log, (B) late log, and (C) stationary phases, and about
105 CFU/mL bacteria were inoculated into LB containing different concentrations of CM. E. coli O157:H7 were grown to early-log (D), late-log (E)
and stationary (F) phase, and about 5 × 108 CFU/mL bacteria were inoculated into LB containing different concentrations of CM. Data are means
± SEM of three independent experiments.
different growth phases was not significant, but CM killed E.
coli O157:H7 efficiently regardless of the bacterial growth
phase.
Antimicrobial Activity of CM in Media. It has been
suggested that chitosan may bind to nutrients, including fat,20
protein,21 and salts,22 in the media that decreases its
antimicrobial activity. Since CM were generated from chitosan,
we evaluated if the antimicrobial activity of CM might be
reduced by nutrients by measuring the antimicrobial activity in
nutrient rich media, Luria−Bertani broth (LB). In addition,
when CM are administered to treat diseases, they would
encounter a complex cellular matrix within the host, therefore
evaluation of the antimicrobial activity in LB media would give
us more realistic insights for CM activity. The antimicrobial
activity of CM was measured at different growth phases with
different numbers of E. coli O157:H7 as measured in isotonic
water.
As shown in Figure 5, it is clear that the antimicrobial activity
of CM is decreased in the presence of nutrients, but CM still
kill bacteria efficiently. At 5 × 105 CFU/mL, the minimum
concentrations of CM needed to completely eliminate E. coli
O157:H7 at early-log, late-log or stationary phase were 0.1, 0.2,
or 0.2% (Figure 5A−C), respectively. The concentrations of
CM in LB media are about 100 times more than that of CM
required to kill the pathogen in isotonic water (Figure 4). This
observation was repeated with higher numbers of E. coli
O157:H7. At 5 × 108 CFU/mL, about 50−100 times more CM
were needed to kill pathogens completely in LB media
compared to isotonic water. CM at 0.2% could eliminate
early and late log phase pathogen within 12 h incubation
(Figure 5D,E), while 0.4% of CM was required to kill E. coli
O157:H7 at stationary phase (Figure 5F). Taken together, CM
exert significant antimicrobial properties against E. coli
O157:H7 at all growth phases, although the antimicrobial
activity is significantly diminished in the presence of nutrients,
probably due to nonspecific binding to nutrients that might
limit available CM to attack pathogens.
CM Kill Pathogens in the Simulated Gastrointestinal
Tract Conditions. The antimicrobial activity of CM was
evaluated mimicking the conditions in which orally administered CM pass through the gastrointestinal tract. Thus, we
prepared synthetic gastrointestinal fluids to simulate real gastric
and ileal intestinal environments. The pH of synthetic gastric
fluid was adjusted to 1.5 or 2.5 to simulate the normal range of
the stomach (or abomasum in ruminants). In the natural
environment, the bacteria obtained by animals are at stationary
phase, so we inoculated bacteria at stationary phase to the
synthetic gastric fluids at 5 × 108 CFU/mL. At pH 1.5, all
bacteria were eliminated after 1 h due to the severe acidic
environment (data not shown), so the log reduction of E. coli
O157:H7 was tested after 0.5 h of incubation (Figure 6A).
Only trace amount of CM (0.00001%) was necessary to kill the
pathogen. A 6-log reduction was obtained by 0.000005% of CM
in the stomach at pH 1.5, indicating antimicrobial activity of
CM is significantly enhanced at lower pH. When the pH was
increased to 2.5, 0.1, 0.001, and 0.0001% CM could inhibit the
bacteria completely after 0.5, 1, and 2 h, respectively (Figure
6B). These data indicate that CM will kill pathogens from the
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Figure 6. Antimicrobial activity of CM in synthetic gastrointestinal
fluids. About 5 × 108 CFU/mL E. coli O157:H7 were inoculated into
the fluids. (A) Log reduction of bacteria after 0.5 h in synthetic gastric
fluid (pH = 1.5) containing 0 to 0.00002% CM. Data are means ±
SEM of three independent experiments. Means with different letters
differ (P < 0.05). (B) Survival curve of bacteria in synthetic gastric fluid
(pH = 2.5) containing 0 to 0.1% CM. (C) Survival curve of bacteria in
synthetic small intestinal fluid containing 0 to 0.6% CM.
Figure 7. In vitro ruminal fermentation assay. (A) In vitro true
digestibility (%) of substrate in CM treatments. (B) pH of rumen
fluids after 24 h in vitro digestion. (C) Total volatile fatty acid (mM)
after 24 h in vitro digestion. No statistical difference was detected
among treatments (P > 0.05).
subclinical acidosis in cattle, can reduce the growth and
performance of ruminants. In the assay, no statistical difference
in pH was detected among treatments (Figure 7B). The
similarity of in vitro true digestibility and pH of CM-treated
and control samples indicated that CM did not disrupt the
normal function of the rumen. As shown in Figure 7C, the total
volatile fatty acid (VFA) of all the CM treatments did not have
a difference compared to the control. Similarly, adding different
concentrations of CM did not change the production of any
VFA compared to the control (Table 2). Overall, no linear,
quadratic or cubic regression was associated with the
treatments. Taken together, CM do not alter rumen function,
indicating CM may not cause adverse side effects, at least under
the tested parameters.
stomach to a great extent, therefore smaller amounts of E. coli
O157:H7 can pass into the small intestine if CM are orally
administered. Next, we examined the antimicrobial activity of
CM in ileal fluid with 5 × 104 CFU/mL bacteria at stationary
phase (Figure 6C). In the simulated small intestinal environment, 0.6% CM decreased the bacteria to nondetectable
concentrations within 6 h, whereas 0.4% CM took 12 h to reach
complete inhibition of the bacteria. These data suggest that CM
are able to eliminate pathogens at a low concentration in
different organs when they are orally administered and have
great potential to increase human and animal health through
the prevention and treatment of bacterial infections.
In Vitro Ruminal Digestibility Assay. A risk assessment
of CM would provide us with a comprehensive and direct
understanding of CM that may cause unnecessary health
consequences during oral administration. We conducted a risk
assessment of CM with an in vitro ruminal digestibility assay to
estimate how CM affect the normal function of the rumen. As
shown in Figure 7A, the in vitro true digestibility of CM
treatments (0.2, 0.4, and 0.6% of substrate) did not differ from
the control after 24 h incubation. In addition, pH change of the
rumen fluid after digestion serves as an indicator of rumen
function alteration. If pH decreases to less than 5, the rumen
microflora cannot function normally due to acute ruminal
acidosis. Even a sustained pH of 5.5−5.8, which is indicative of
■
DISCUSSION
Our findings reveal the potential of CM, driven from natural
biopolymer chitosan, to treat infectious diseases caused by
antibiotic-resistant microorganisms. CM have broad-spectrum
antimicrobial activity without raising mutation rates when
treated at sublethal levels. Furthermore, risk assessment of CM
revealed by the normal function of the rumen indicates that
CM unlikely cause side effects.
According to the Centers for Disease Control and
Prevention’s report, Antibiotic Resistance threats in the United
States, 2013, nearly 23 000 people each year are killed by
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Table 2. Effect of CM on Volatile Fatty Acid Concentrations (mM) after 24 h in Vitro Ruminal Digestion
CM (% of substrate)a
a
contrast P-valueb
item
0
0.2
0.4
0.6
SEM
L
Q
C
acetate
propionate
isobutyrate
butyrate
isovalerate
valerate
A:P
67.28
36.87
8.29
21.10
5.38
20.81
2.10
54.10
26.70
5.76
13.18
4.31
13.02
2.03
57.92
28.21
7.02
15.33
3.74
14.44
2.09
56.34
25.72
5.57
13.71
4.23
13.83
2.24
14.88
8.59
1.54
4.32
0.62
4.17
0.11
0.28
0.26
0.18
0.17
0.33
0.12
0.20
0.36
0.54
0.63
0.32
0.77
0.12
0.17
0.54
0.57
0.21
0.33
0.24
0.82
0.87
No statistical difference was detected (P > 0.05). bL, linear; Q, quadratic; C, cubic.
ARMs.23 The number of antibiotic-resistant microbes is
increasing and will continue to increase due to the slow
development of new antibiotics and lack of alternative therapies
for infectious diseases. Recently, Ling et al.5 reported a
potential 11 amino acid peptide antibiotic, named teixobactin,
produced by a soil microorganism that was cultured by isolation
chip, which allowed the culture of microorganisms that had not
previously been able to be cultured in vitro. This finding is
especially important because the antibiotic was not only
isolated from previously identified nonculturable bacteria but
also has broad-spectrum antimicrobial activity that also does
not raise antibiotic resistance.5 CM were similar to teixobactin
in terms of broad-spectrum of antimicrobial activity (Figure 1)
without raising resistance (Figure 2B) that did not induce an
increase in mutation when incubated with CM at MIC levels
for 15 days while ampicillin, tetracycline, and polyminxin B did
within a period of 4 days. Compared with teixobactin, CM
harbor great antimicrobial activity against both Gram-positive
and Gram-negative bacteria, whereas teixobactin showed
limited antimicrobial activity against Gram-negative bacteria.
It is plausible that CM exert great antimicrobial activity due to
broader targets, including OmpA and LPS in Gram-negative
bacteria but teichoic acid in Gram-positive bacteria.
Mutation rate is an important indicator of the adaption speed
of antibiotic resistance. 24 It is believed that cationic
antimicrobial peptides (AMPs) have lower mutation rates
because of their mode of action that break bacterial membranes,
compared to other antibiotics such as ampicillin, ciprofloxacine,
and kanamycin that increase the mutation rate significantly.25
Like AMPs, CM did not increase mutation rate similarly to the
negative control (Figure 2A). In addition, CM break bacterial
cell walls shown by live/dead assay stained with propidium
iodide, suggesting CM may have a similar mode of action like
AMPs to kill pathogens. It has been shown that antibiotic
treatments at sublethal levels can lead to multidrug resistance
via radical oxygen species (ROS) formation in the presence of
oxygen19 which raised concerns for prophylactic use of
antibiotics routinely fed to food-producing animals to promote
growth. As shown in Figure 3, the increased MIC was not
observed when 0.5× MIC CM were treated to kill pathogens,
suggesting CM treatment at sublethal levels may not cause
multidrug resistance, even if CM are used for prophylactic
purpose in animals that may reduce the use of medically
important antibiotics in animals.
Only 0.002% CM was needed to eliminate 5 × 105 CFU/mL
E. coli O157:H7 in isotonic water, which is 100 fold less than
the concentration needed in LB broth (Figure 4 and 5). In
addition, it is noticeable that in isotonic water, the MBC levels
for each bacteria were 50−80 000 fold lower than the MIC in
Mueller Hinton Broth (MHB). It could be due to the
nonspecific binding of CM to nutrients in complex media
including carbohydrates, beef infusion solids, and casein
hydrolysates that may bind to CM to block binding to target
cells. Nutrients that may bind to CM have not been identified
in this study, but it would be beneficial to maintain strong
antimicrobial activity observed in isotonic water, which is free
of potential blocking materials, to develop alternative
antimicrobial agents in future studies. Antimicrobial activity
of CM was concentration dependent (Figures 4 and 5) as
antimicrobial activity was increased when more CM were added
to the cultures. However, when we mimicked the late stage of
bacterial infection by increasing cell numbers to 5 × 108 CFU/
mL, only 2 times more CM were needed to kill 1000 times
more bacteria (Figure 4D,E) within 12 h. On the basis of these
data, we speculate that CM binding to target cells are reversible
and CM might be released after killing pathogens. It has been
shown that chitosan aggregates with bacterial cells, which is not
reversible, to kill them,26 therefore CM may have different
antimicrobial activity different from chitosan. This hypothesis
might be consistent with the previous finding that CM bind to
OmpA by hydrophobic interaction rather than ionic
interaction,14 by which chitosan disrupts cell walls.
In this study, we evaluated the in vitro antimicrobial activity
of CM in simulated gastrointestinal environments to understand the fate of CM when they are orally administered. As it is
required to kill pathogens in the large intestinal tract where E.
coli O157:H7 primarily colonize, CM should maintain the
antimicrobial property without being disrupted or degraded by
bile salts or digestive enzymes in the gastrointestinal tract. In
synthetic gastric fluid at pH 1.5 or 2.5, mimicking the pH of the
stomach before or after ingesta enters, less than 0.00001% of
CM at pH 1.5 was sufficient to eliminate 5 × 108 CFU/mL cells
within 30 min (Figure 6A). This observation is consistent with
the previous report that the bactericidal effect of CM is
enhanced in acidic conditions.14 Jeong et al.15 reported that
CM administered orally with feed reduced E. coli O157:H7
shedding significantly in a study with calves in a crossover
design. In the study, feeding CM for 6 days after inoculation
with 106 CFU of E. coli O157:H7 significantly reduced the total
number of E. coli O157:H7 and shortened the duration of
shedding from 13.8 days to 3.8 days. However, the mode of
action for the reduced shedding was not explained. We have
shown that CM exert enhanced antimicrobial activity in the
stomach and maintains the activity in the intestinal tract
(Figure 6A−C) in simulated environments, explaining that the
previously unknown mechanisms of reduced shedding was
probably mediated by antimicrobial activity of CM in the
gastrointestinal tract.
Due to the cellular and environmental toxicity, nanoparticles
(NP) have not been extensively applied in human and animal
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Research Article
ACS Applied Materials & Interfaces
clinics,27 although they provide great potential as an alternative
treatment option.28 We conducted risk analysis to evaluate
adverse side effects of CM by using an in vitro animal model
and a ruminal digestibility assay. For ruminants, maintaining the
normal functions of microflora in the rumen is critical for
digestion and absorption.29 This is because ruminal microbes
are primarily responsible for digestion of food and the fatty
acids they produce account for a significant proportion of the
energy required for maintenance and animal growth.29 If the
homeostasis of the rumen is disrupted, ruminal dysfunction will
occur and is correlated with death in severe cases.30 Therefore,
measuring rumen function could be a sensitive indicator for risk
assessment. As shown in Figure 7, the concentrations of CM
ranging from 0.2 to 0.6% did not alter in vitro true digestibility,
pH, or total VFA produced after fermentation, indicating CM
have no detectible adverse side effects. Therefore, 0.2% CM,
which was shown to effectively remove E. coli O157:H7 in the
gastrointestinal tract, may be safe to decrease pathogen
concentration without disrupting the normal rumen microflora.
The commonly and extensively fed antibiotic, monensin, for
prophylactic purpose in beef cattle is reported to decrease dry
matter digestibility both in vitro and in vivo.31,32 In contrast to
monensin, CM did not alter digestibility. Therefore, it is clear
that CM are biocompatible without recognized adverse side
effects such as cell toxicity that are typically observed with
metallic nanoparticles.8,33
dilution decreased 99.9% of the initial bacterial concentration. MIC
was determined by broth macrodilution according to CLSI guidelines.35 MBC was determined in isotonic water (0.264 M glucose
solution). Bacterial concentrations were adjusted to approximately 5 ×
105 CFU/mL.
Determination of Mutation Rate. The mutation rate experiment
was conducted using a rifampicin-based selection method.36 An
overnight culture of E. coli O157:H7 was diluted 1:10 000 into 50 mL
LB (Difco, BD & Co., East Rutherford, NJ) and grown for 3.5 h at 37
°C. The culture was then diluted 1:3 into fresh LB containing no
antibiotic (negative control), 4 μg/mL ampicillin (0.25× MIC), 8 μg/
mL ampicillin (0.5× MIC), 0.05% CM (0.25× MIC), or 0.1% CM
(0.5× MIC), respectively. Ten 1 mL replicates of each treatment were
grown for 24 h at 37 °C. The cultures were serial diluted and plated on
LB agar plates, containing 100 μg/mL rifampicin, and then the plates
were incubated for 48 h at 37 °C. Colonies were counted to determine
CFU/mL. The mutagenesis rate was calculated by the MSS maximumlikelihood method using the online web tool FALCOR (http://www.
mitochondria.org/protocols/FALCOR.html).16
Resistance Acquisition. To determine if sublethal amounts of
CM can increase MIC, E. coli O157:H7 cultures were grown in 1 mL
of MHB containing 0.25×, 0.5×, 1×, 2×, and 4× MIC, respectively.
After 24 h, bacterial concentration was measured to determine the
level of bacterial growth. Cultures from the second highest
concentrations that allowed growth (OD600 ≥ 2) were diluted 1:100
into fresh MHB containing different concentrations of CM described
above. Cultures that grew above the MIC levels were plated on LB
plates and their MIC levels were determined by broth macrodilution.
Sequential passaging was repeated daily for 15 d. Ampicillin,
tetracycline and polymyxin B were used as controls.
Determination of MIC Variability. E. coli O157:H7 cultures were
grown for 5 d in MHB containing no treatment, 4 μg/mL ampicillin
(positive control), or 0.05% CM, respectively. The bacteria were
diluted 1:100 into fresh MHB containing the respective treatments
daily. Each day thereafter for 5 d, aliquots of the culture were used to
measure MICs for ampicillin, tetracycline, and kanamycin.
Synthetic Gastrointestinal Fluids. The synthetic gastrointestinal
fluids were prepared according to Beumer et al.37 The simulated
stomach environment was made by adding proteose-peptone (8.3 g/
L), D-glucose (3.5 g/L), NaCI (2.05 g/L), KH2PO4 (0.6 g/L), CaCI2
(0.11 g/L), KCI (0.37 g/L), porcine bile (0.05 g/L), lysozyme (0.1 g/
L), and pepsin (13.3 mg/L). The pH was adjusted to 1.5 and 2.5 with
HCl. The simulated ileal environment was prepared by adding
proteose-peptone (5.7 g/L), D-glucose (2.4 g/L), NaCI (6.14 g/L),
KH2PO4 (0.68 g/L), NaH2PO4 (0.3 g/L), NaHCO3 (1.01 g/L),
porcine bile (5.6 g/I), lysozyme (0.2 g/L), α-amylase (1000 U/l),
lipase (960 U/l), trypsin (110 U/l), and chymotrypsin (380 U/l). The
pH was adjusted to 7.0.
Antimicrobial Activity Assay. A single colony of E. coli O157:H7
was inoculated in 5 mL of LB and incubated at 37 °C with shaking at
200 rpm overnight. The next day, the culture was diluted 1:100 in
fresh LB and again incubated at 37 °C. For antimicrobial activity, the
bacteria were grown until reaching early-log (OD600 = 0.5), late log
(OD600 = 1.0), or stationary (OD600 = 3.0) phase. Approximately 5 ×
104 or 5 × 108 CFU/mL of bacteria were inoculated into 2 mL of LB
or isotonic water containing different concentrations of CM. The
cultures were serial diluted and plated on LB agar at 0, 2, 4, 6, 12, and
24 h. The plates were incubated at 37 °C overnight to count CFU. In
synthetic gastrointestinal fluids, 5 × 108 CFU/mL of bacteria were
inoculated initially.
In Vitro Ruminal Digestibility. The ruminal fluid was
representatively collected from 2 nonlactating, nonpregnant, ruminally
cannulated Holstein cows 3 h after consuming a ration of 50%
bermudagrass hay and 50% concentrate containing corn (73.75%),
cottonseed hull (7.5%), corn gluten feed (10%), oil (0.75%), calcium
carbonate (0.5%), and a commercially produced protein pellet
(Jacko52, 7.5%, DM basis). Standard practices of animal care and
use were applied to animals used in this project. Research protocols,
including permission for cannulated Holstein cows and collection of
ruminal fluid, were approved by the University of Florida Institutional
■
CONCLUSIONS
Our findings suggest that CM can be a promising therapeutic
candidate for treatment of infections caused by antibioticresistant bacterial pathogens. CM do not increase mutation rate
and cause resistance in a short time frame as well as harbor
strong antimicrobial activity in different environments,
mimicking the real world situations. The results of our study
show great potential of CM to treat infectious diseases caused
by especially multidrug resistant microorganisms without
adverse side effects.
■
MATERIALS AND METHODS
Preparation of Chitosan Microparticles. CM were prepared as
described previously34 with minor modifications. A 2% (wt/vol)
chitosan (Molecular weight 50−190 kDa, deacetylation degree 75−
85%, 448869−250G, Sigma-Aldrich) solution was prepared with 2%
acetic acid (v/v) and 1% tween 80 (v/v). For cross-linking, the
chitosan solution was stirred and 10% of sodium sulfate (w/v) was
added dropwise during 25 min of sonication. The sonication process
was continued for 25 min. The CM were collected by centrifugation
(8200g) and washed with sterile water. The weight of CM was
measured after freeze-drying.
Live/Dead Viability Assay. Bacterial viability was determined
using the Live/dead BacLight Bacterial Viability Kit (Molecular
Probes, Inc., Eugene). Briefly, 5 × 107 colony forming units per
milliliter (CFU/mL) E. coli O157:H7, extended-spectrum betalactamases (ESBLs) producing E. coli, K. pneumonia, methicillinresistant S. aureus, vancomycin-resistant Enterococcus and V. cholerae
O1 El Tor were inoculated into 1 mL of MHB (Difco, BD & Co., East
Rutherford, NJ) containing CM at the MIC level specific for each
bacteria, respectively. Bacterial culture was incubated at 37 °C for 2 h
and then incubated in the dark at ambient temperature with SYTO 9
and propidium iodide for 15 min. Bacteria were observed using the
fluorescence microscope (EVOS XL Cell Imaging System).
Minimum Inhibitory Concentration (MIC) and Minimum
Bactericidal Concentration (MBC). The MIC is defined as the
lowest concentration of CM that prevents visible growth of the
pathogens in susceptibility test by broth dilution. MBC is the first drug
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ACS Applied Materials & Interfaces
Animal Care and Use Committee (IACUC Protocol no. 201508765).
The ruminal fluid was filtered through 4 layers of cheesecloth
immediately after collection. The substrate was the same as the ration.
Concentrations of CM at 0, 0.2, 0.4, and 0.6% of substrate were mixed
with 0.5 g substrate in Ankom bags and put in 100 mL polypropylene
tubes. Goering and Van Soest (1970)38 medium were prewarmed (39
°C) and flushed continuously with CO2 before ruminal fluid addition.
The rumen fluid inoculum and Goering and Van Soest (1970)38
medium (52 mL) were added to each tube and the suspension was
incubated for 24 h at 39 °C. After the incubation, the rumen fluid
inoculum from the in vitro ruminal digestibility was measured for pH
(Accumet Excel XL 25, Fisher Scientific). After measuring the pH, the
fluid inoculum was acidified with 50% H2SO4 (1% v/v of rumen fluid
inoculum), and centrifuged at 8000g for 15 min at 4 °C. The
supernatant was used for VFA analysis. Concentration of VFA was
determined by high performance liquid chromatography (HPLC)
system (Hitachi, L2200, L2130, and L2400; Tokyo, Japan) and a BioRad Aminex HPX-87H column (Bio-Rad Laboratories). The residues
in the Ankom bags were dried at 105 °C for 24 h, weighed, and the in
vitro true digestibility was calculated. The experiment was conducted
with four replicates in two independent trials conducted on different
days.
Statistical Analysis. Data were analyzed using the GLIMMIX
procedure of SAS version 9.1 (SAS Institute Inc., Cary, NC) and a
statistical model that included different concentrations of treatment
was generated. Analysis of means was calculated using the Tukey test.
Multiple regression relationships in VFA profile and CM concentrations were analyzed using the stepwise multiple regression
procedure of SAS. All experiments were conducted in triplicate if
not mentioned above. Significance was declared at P ≤ 0.05.
■
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AUTHOR INFORMATION
Corresponding Author
*E-mail: kcjeong@ufl.edu. Phone: 1-352-294-5376.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors are grateful to S. Markland for helpful discussion
and technical support. This material is based upon work that is
supported by the National Institute of Food and Agriculture,
U.S. Department of Agriculture, under award number 201467021-21597 and 2015-68003-22971 to KCJ.
■
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