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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

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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 10700

DOI: 10.1021/acsami.6b00894 ACS Appl. Mater. Interfaces 2016, 8, 10700−10709

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ACS Applied Materials & Interfaces 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

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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 10701


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ACS Applied Materials & Interfaces 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 10702


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ACS Applied Materials & Interfaces

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 ( 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

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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 10705


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ACS Applied Materials & Interfaces 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 10706


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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

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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.

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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 10707


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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.

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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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