http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.893001
ORIGINAL ARTICLE
Antimicrobial activity of zinc oxide (ZnO) nanoparticle against Klebsiella pneumoniae Lanka Shalini Reddy, Mary Magar Nisha, Mary Joice, and P. N. Shilpa
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Department of Biotechnology, Sathyabama University, Chennai, Tamil Nadu, India
Abstract
Keywords
Context: Zinc oxide nanoparticles (ZnO Nps) have potential application in piezoelectric nanogenerator and in biotechnology. Objective: The antibacterial activity of ZnO Nps on Klebsiella pneumoniae (ATCC 70068) and mode of action of ZnO Nps was investigated. Methods: ZnO Nps was synthesized by a precipitation method and characterized using scanning electron microscopy (SEM) and X-ray diffraction (XRD) methods. In vitro susceptibility of K. pnumoniae of the ZnO Nps was detected using the disk diffusion method, and the minimum inhibitory concentration (MIC) value was determined using the serial dilution method. The chemical and physical interaction between the cell envelope of K. pneumonia and ZnO Nps was investigated. The effect of ZnO Nps on the cytotoxic activities of K. pneumonia was investigated using a HEp-2 cell line. Results: The MIC of ZnO Nps was found in 40 mg/ml. The standard growth curve showed that ZnO Nps of 0.75 mM inhibited K. pneumoniae after 4 h. The interaction with outer membrane protein (OMP) and lipoploysacharride (LPS) residues showed modulation in 66 kDa and 29 kDa proteins with the use of increasing concentrations of ZnO Nps. The amount of nucleic acid and protein released from the cells increased with the ZnO Nps concentration used. Importantly, the OD of the ZnO Nps-treated cells decreased within 30 min of incubation in the presence of SDS. ZnO Nps-treated K. pneumoniae were five-fold less infectious in the HEp-2 cell line at doses between 0.50 and 0.75 mM. Discussion: These results suggest the potential antibacterial use of ZnO Nps against K. pneumoniae infections.
Agar diffusion assay, growth curve, HEp-2 cells, lipoploysaccharride, metal oxide nanoparticle, minimum inhibitory concentration, outer membrane protein
Introduction The treatment of bacterial infections is increasingly complicated by the ability of bacteria to develop resistance to antimicrobial agents. There are several new strategies that have been employed to control microbial infection. Metal oxide nanoparticles, a new class of materials, are increasingly being recognized for potential use in research and healthrelated application. Recent studies have demonstrated that specially formulated metal oxides Nps possess good antibacterial potential (Azam et al., 2012) and Nps-based antimicrobial formulations could be used as an efficient bactericidal material in modern medicine (Padmavathy & Vijayaraghavan, 2008). Among several metal oxides, ZnO has recently achieved special attention regarding potential electronic application due to its unique optical, electrical, and chemical properties (Jiang et al., 2009). ZnO appears to be strongly resisted to microorganisms where as ZnO Nps exhibit strong antibacterial activities on broad-spectrum pathogenic bacteria Correspondence: Dr. P. N. Shilpa, Virtis Bio Labs, 34, Velautham Gounder Complex, Dharman Nagar, Ammapalayam Main Road, Salem 636 005, Tamil Nadu, India. Tel: +91 9597926827. E-mail: [email protected]
History Received 9 March 2013 Revised 28 December 2013 Accepted 6 February 2014 Published online 15 July 2014
such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, E. coli O157:H7, Salmonella enteritidis, Salmonella typhimurium, Pseudomonas fluorescens, and Listeria monocytogenes (Adams et al., 2006; Huang et al., 2008; Jones et al., 2008; Ravishankar Rai & Jamuna Bai, 2011). ZnO nanoparticles (12 nm) inhibited the growth of E. coli by disintegrating the cell membrane and increasing the membrane permeability (Huh & Kwon, 2011). Recent lines of evidence suggest that the synthesis of hydrogen peroxide (Ravishankar Rai & Jamuna Bai, 2011) and eventual penetration of cell envelope and disorganization of bacterial membrane following contact with ZnO Nps are the two potential mechanisms linked to bacterial inhibition (Brayner et al., 2006; Huang et al., 2008). However, very few detailed studies were conducted on the relationship between the ZnO Np and the antibacterial activity. Klebsiella pneumoniae is an important pathogen frequently implicated in respiratory and urinary tract infections (UTI) of hospitalized patients. It is a part of the respiratory and intestinal microbiota of humans and is isolated from the oropharyngeal cavity at a frequency of 1–6% (Mohammed et al., 2010). It is also strongly associated with infections namely septicemias, and iatrogenic infections, since the
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hospital environment is associated with long-staying hospitalized patients and/or those who have been in the intensive care unit (ICU), under oropharyngeal intubation. Between half and three-quarters of all bacteremias caused by K. pneumoniae are nosocomial and are resistant to ampicillin and carbenicillin, albeit being frequently susceptible to cephalosporins, cotrimoxazole, aminoglycosides, and imipenem (Archana & Harsha, 2011). The growth inhibition of K. pneumoniae has been tested using several inorganic nano-metals by others (Ravishankar Rai & Jamuna Bai, 2011). Nonetheless, ZnO showed greater bacterial growth inhibition as a result of potential intracellular incorporation of Nps (Jones et al., 2008). Notwithstanding the effectiveness of ZnO as an antimicrobial agent, questions remain as to whether bacteria will develop resistance to ZnO (similar to that seen with modern antibiotics). Numerous models have been suggested including deactivation of certain bacterial enzymes, disruption of gene replication, and limitation of cellular membrane function (Mohammed et al., 2010). However, the true mechanism for ZnO antibacterial action remains uncertain. The cell wall of most pathogenic bacteria is composed of complex surface proteins for adhesion and colonization, and components such as polysaccharides and teichoic acid that protect against host defenses and environmental conditions (Katsikogianni & Missirlis, 2004). These components are charged macromolecules, and, therefore, specific interactions to disrupt their main functions and location may be triggered by introducing specific groups on the surface of the microorganism. Hence, we set out to investigate the chemical and physical interaction between the cell envelope of K. pneumonia and ZnO Nps. Further, investigations were also undertaken to investigate the effect of ZnO Nps on the cytotoxic activities of K. pneumonia using a HEp-2 cell line.
Materials and methods Bacteria Standard strain of Klebsiella pneumoniae (ATCC 70068) was collected from Global Hospital, Chennai, India. For routine use, the cultures were maintained on nutrient agar (NA) (Hi-media, Mumbai, India) plates/slants. Glycerol stocks were prepared and placed at 20 C for long-term storage. Cell line Human esopharyngeal carcinoma cell line (HEp-2) was purchased from KIPM (King Institute of Preventive Medicine, Chennai-32, India), and grown in minimal essential media supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 50 IU/ml penicillin, and 50 mg/ml streptomycin, incubated at 37 C under humidified atmosphere with 5% CO2. ZnO nanoparticles ZnO Nps was synthesized by a standard method (Padmavathy & Vijayaraghavan, 2008). This was also used to investigate the effect of ZnO Nps on the bacterial samples held at varying concentrations 0.25, 0.50, and 0.75 mM by performing a series of other investigations described by Archana and Harsha (2011).
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Characterization of ZnO Nps Phase purity and grain size were determined by XRD analyses recorded on a Siefert X-ray diffractometer (Siefert, Germany) ˚ ) at 60 keV over the using CuKa radiation (l ¼ 1.54016 A range of 20–70 . The average grain size was determined by X-ray line broadening using the Scherrer equation after incorporating corrections. The morphology and size of the particle were determined using a SEM (Hitachi S-4500, Hitachi, Shiga, Japan). MIC and growth curve Test organism K. pneumoniae were grown separately in a 50 ml sterilized nutrient broth medium and kept in a shaker incubator at 37 C for 14 h (overnight incubation). On the subsequent day, test organism cultures were transferred at the rate of 1% in 100 ml nutrient broth. MIC assay of ZnO Nps The initial concentration (8 mg/ml) was diluted using double fold serial dilution by transferring 5 ml of the sterile ZnO Nps (stock solution) into 5 ml of sterile nutrient broth to obtain 4 mg/ml concentration. The above process was repeated several times to obtain other dilutions. To different concentrations of ZnO Nps, 0.1 ml of the standardized bacterial cell suspension was added and incubated at 37 C for 24 h. The growth of the inoculums in the broth is indicated by turbidity or cloudiness of the broth and the lowest concentration of the ZnO Nps, which inhibited the growth of the test organism, were taken as the minimum inhibitory concentration (MIC). Growth curve Various concentrations of ZnO Nps (0.25, 0.50, and 0.75 mM) were carefully placed into each flask, leaving one as a control to track the normal growth of the microbial cells without nanoparticles. Experiments were performed using both a negative control (flask containing cells plus media) and a positive control (flask containing nanoparticles plus media). The flasks were shaken at 180 rpm at 37 C in a shaker incubator. Optical density measurements from each flask were taken every 1 h to record the growth of the microbes in a spectrophotometer set at 600 nm. The growth rate of microbial cells interacting with the nanoparticles was determined from a plot of the log of the optical density versus time. Protein fractionation Outer membrane proteins (OMPs) were isolated from overnight cultures of K. pneumoniae that have been incubated with 0.25, 0.50, and 0.75 mM of ZnO Nps and centrifuged for 5 min at 7000 g. Pellets were washed once with 20 mM Tris, 10 mM EDTA, pH 8 (TE), and resuspended in the same buffer. Bacteria were disrupted by sonication for 1 min, followed by 2 min blank, and an additional 1 min sonication. Samples were centrifuged for 5 min at 7000 g to remove debris, and the resulting supernatant was centrifuged for 1 h at 60 000 g at 4 C. The clear supernatant was retained as cytosolic fraction. The pellet was resuspended in TE buffer and the protein concentration was estimated. Protein concentration was adjusted to 5 mg/ml and solubilizes with sodium
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DOI: 10.3109/13880209.2014.893001
lauryl sarcosinate 1% w/v (final concentration) at 4 C for 1 h. Samples were centrifuged again for 1 h at 60 000 g at 4 C and the supernatant was taken for analysis of inner membrane fractions. The pellet containing OMP was resuspended in 1% SDS and boiled for 10 min. The resulting sample was run on an SDS-PAGE unit (Genei, Bangalore, India) by adding 5 ml of loading dye to the samples. Following electrophoresis, the gel was stained in commassie brilliant blue (CBB) for overnight and was destained to view the protein bands using a transilluminator (Bharathi et al., 2008).
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LPS extraction LPS was extracted by a standard method (Galanoso et al., 1969). Briefly, the overnight culture of bacterial suspension (108 CFU units/ml) incubated with 0.25, 0.50, and 0.75 mM ZnO Nps was centrifuged at 10 000 g. The pellet was washed with PBS (pH 7.2) and resuspended in water. Subsequently, equal volumes of hot (65–70 C) phenol was added and stirred vigorously. The suspension was brought to room temperature and centrifuged at 8500 g. The supernatant was transferred to a conical centrifuge tube. Phenol was re-extracted from the preparation and aqueous solution was pooled. Sodium acetate (0.5 M) and 10 volume of 95% ethanol were added, and placed overnight at 20 C in order to precipitate the LPS. Following a brief centrifugation at 2000 g at 4 C, the final pellet was suspended in d.H2O and the final precipitate was used in SDS-PAGE. Protein leakage analysis Protein leakage analysis was performed using a standard Bradford assay (Bradford, 1967). Briefly, K. pneumoniae cells were treated with 0.25, 0.50, and 0.75 mM ZnO Nps in nutrient broth (pH 7.4) for 1 h and 5 h at 37 C. Supernatant was collected after centrifugation (6000 rpm) for 15 min. For each sample, 200 ml of the supernatant was mixed in 800 ml of the Bradford reagent. The optical density (595 nm) was measured after 10 min of incubation in the dark. BSA was used as a standard protein. Membrane stability of K. pneumoniae The effect of ZnO Nps on the outer membrane of the bacteria was checked by SDS treatment of pre-treated cells (with ZnO Nps) of K. pneumoniae. Active culture of K. pneumoniae was mixed in PBS-buffer containing 0.25, 0.50, and 0.75 mM ZnO Nps and incubated for 30 min. Cells were centrifuged and the pellet was mixed in the same volume of PBS. SDS (0.15%) was mixed in ZnO Nps-treated cells. OD was measured every 2 min. Nucleic acid analysis K. pneumoniae cells were treated with 0.25, 0.50, and 0.75 mM ZnO Nps in nutrient broth (pH 7.4) at 37 C. Supernatant was collected after centrifugation (6000 rpm) for 15 min. The amount of nucleic acid released in 0.25, 0.50, and 0.75 mM ZnO Nps-treated cells was measured at 260 nm using a UV–VIS spectrophotometer. For preliminary identification of nucleic acid leakage, ninhydrin test showed a positive result. The experiment was repeated for minimizing error (Khalil & Villota, 1988).
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Isolation of catalase Cell extracts of bacteria incubated with 0.25, 0.50, and 0.75 mM were collected at the mid-logarithmic or stationary phase by centrifugation at 10 000 g for 5 min at 4 C. Pellets were washed once, resuspended in 200 ml of electrophoresis buffer, and sonicated twice in an ice water bath for 30 s with a sonicator. The sonicate was then centrifuged at 15 000 g for 20 min at 4 C and native PAGE (10%) was run. The gel was soaked in d.H2O to remove trace of running buffer. The gel was soaked in 200 ml of 3 mM solution of H2O2 for 10 min, rinsed in d.H2O and incubated in 1% potassium ferric cyanide for 10 min in the dark. Cytotoxicity assay HEp-2 cells were grown in MEM supplemented with 5% FCS, 2 mM L-glutamine, and 50 IU/ml penicillin and 50 mg/ml streptomycin for 3–4 d in a CO2 incubator. HEp-2 cells were grown to confluence in 96-well plates. The confluent monolayer was loaded with different concentrations of ZnO Nps and equal multiplicity of infection (MOI) (50:1) of K. pneumoniae. The percentage of cytotoxicity was assessed by MTT as well as lactate dehydrogenase (LDH) assay (Das Niranjali, 2008).
Results The ZnO Nps was synthesized by the precipitation and surface modification methods. The XRD patterns of ZnO samples prepared by the two methods are shown in Figure 1. Figure 1(a) shows the peak broadening of the XRD pattern for the precipitation method. With increasing annealing temperature, the crystalline nature of the sample appears to improve and the grain size increased. The broadened peaks in the XRD pattern obtained by surface modification (Figure 1b) indicate the formation of ZnO nanocrystals with small crystallites. The crystallite size obtained by the two synthesis methods was estimated by the Scherrer equation and found to be in the range of 20–40 nm. Characterization of ZnO Nps was confirmed by SEM analysis where it shows the variations in particle size of 159 nm in the precipitation method and 88.7 nm of reduced size by the surface modification method. The particle interaction and aggregation are observed in ZnO Np obtained by the precipitation method. The presence of capping molecules appears to affect the kinetics of nucleation and accumulation in such a way that the rate of growth of large particle decreases while that of small particles remains the same. This resulted in a narrowing of the size change of the particles after the addition of a capping molecule. The reduction in size was observed by surface modification method than compared with the precipitation method (Figure 2a and b). The representative results of the bacterial count for different concentrations for ZnO Nps for K. pneumonia is shown in Figure 3. By doing serial dilution, ZnO Nps at different concentrations showed a dramatic decrease in the number of colonies formed. Serially with decreasing concentrations of Nps, there will be an increase in the bacterial growth. Thus, with increased Nps concentration, greater antibacterial effect was observed. Figure 4 show the effect of
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Figure 1. (a) XRD pattern of ZnO Nps formed by precipitation method. (b) XRD pattern of ZnO Nps after surface modification.
Figure 2. (a) Characterization of ZnO Nps synthesized by the precipitation method using SEM analysis. (b) Modification of ZnO Nps by the surface modification method using SEM analysis.
ZnO NPS on the growth of K. pneumoniae where timedependent changes in bacterial growth are monitored by measuring OD at 595 nm. The OD at 595 nm is due to the scattering of light by the bacterial cells. It is a function of bacterial cell density and thus correlates with the growth of
the colonies. It is clear that ZnO Nps at increasing concentration decreases the growth of K. pneumoniae. At concentration 0.75 mM, ZnO NPs showed potential inhibition after 4 h as compared with the standard growth curve of K. pneumoniae.
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Figure 3. Minimum inhibitory concentration test of ZnO Nps against K. pneumoniae. Values represent mean ± SD of triplicate experiments.
Figure 4. Growth curve of K. pneumoniae treated with ZnO Nps.
K. pneumoniae was grown overnight at 37 C in medium with and without varied concentrations of ZnO Nps (0.25, 0.50, and 0.75 mM). The proteins were fractioned and subjected to SDS-PAGE. Figures 5 and 6 show the effect of the different concentrations of ZnO Nps on the K. pneumoniae OMP and inner membrane fractions. In the presence of ZnO Nps, the outer membrane as well as the inner membrane fractions showed significant inhibition at higher levels of 66 kDa and lower levels of 29 kDa relative to K. pneumonia grown in the absence of ZnO Nps. In addition, there were some other minor differences between protein
profiles of the bacteria grown in the presence and absence of ZnO Nps. There was a significant diminishing of protein bands (Figure 7) after silver staining. The ZnO Nps showed its maximum effect by direct interaction with the LPS, thereby reducing the virulence of the pathogen. The LPS was degraded in a dose-dependent manner. The amount of nucleic acid released into the suspension was analyzed by the measuring absorbance at 260 nm. The amount of protein released into suspension of treated cells was estimated by the Bradford assay. The amount of protein (Figure 8) and nucleic
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Figure 5. Effect of ZnO Nps on the outer membrane protein of K. pneumoniae. Lane 1: control, Lane 2: 0.25 mM ZnO Nps, Lane 3: 0.5 mM ZnO Nps, Lane 4: 0.75 mM ZnO Nps.
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suspension, no significant decrease in OD was observed after 30 min, either in the presence or absence of ZnO Nps. The result validates the fact that most of the cells inactivated by ZnO Nps remain unlysed in cell suspension in the absence of SDS, and these are highly sensitive to lysis by SDS. It is clear from Figure 10 that the treatment of Nps destabilizes the outer membrane. Inhibition of catalase activity was observed with an increase in the concentration of ZnO Nps showing the iota of oxidative stress induced against the bacteria (Figure 11). In the present study, K. pneumonia-induced cytotoxicity and the protection, if any rendered by ZnO Nps, were determined by two methods, MTT assay and LDH leakage. We observed that the cytotoxicity was more of the same MOI in LDH assay than MTT assay. LDH assay determines the total enzyme leakage by the defoliated as well as the adhered cells in a tissue culture plate, whereas MTT assay measures only the adhered viable cells. In MTT (Figure 12) and LDH assays (Figure 13) the maximum cytotoxicity was observed in cells infected with wild bacteria. The cytotoxicity was almost a half and five-fold less for cells infected with ZnO Npstreated bacteria at a dose of 0.50 mM and 0.75 mM, respectively.
Discussion
Figure 6. Effect of ZnO Nps on the inner membrane protein of K. pneumoniae. Lane 1: control, Lane 2: 0.25 mM ZnO Nps, Lane 3: 0.5 mM ZnO Nps, Lane 4: 0.75 mM ZnO Nps.
Figure 7. Effect of ZnO Nps on lipoploysacharride of K. pneumoniae. Lane 1: control, Lane 2: 0.25 mM ZnO Nps, Lane 3: 0.5 mM ZnO Nps, Lane 4: 0.75 mM ZnO Nps.
acid (Figure 9) released from the cells was directly proportional to the concentration of ZnO Nps used. These results indicate that most of the Nps-treated cells were ghost cells, which released intracellular material into the cell suspension. The OD of ZnO Nps-treated cells reduced dramatically within 30 min of incubation in the presence of SDS; but it did not decrease significantly in the absence of SDS. In untreated cell
The indiscriminate use of antibiotics in the modern society has led to evolution of novel pathogenic antibiotic-resistant bacterial strains. K. pneumoniae is an important human pathogen that has been associated in recent decades with nosocomial outbreaks. Following the use of extendedspectrum cephalosporins, extended-spectrum b-lactamase (ESBL)-producing K. pneumoniae has become an increasingly serious problem worldwide (Hickling et al., 2004; Schurr et al., 2005). Recent evidence suggests that an antibiotic formulation in the form of Nps could be a highly effective bactericidal agent (Huh & Kwon, 2011). This would provide a solution to the increasingly urgent problem of antibiotic resistance, reducing the risk from infections and related complications, which take a heavy toll on vulnerable hospital patients. Outbreaks of antibiotic-resistance bacteria fuel incentives to develop newer effective bactericidal agent (Mohammed et al., 2010). The experimental results so far suggest the possible antimicrobial mechanisms of suspensions of ZnO particles: physical mechanism by attaching onto the bacterial cell walls directly, biological mechanism by interacting with the cell membrane components and chemical mechanism by producing active species (Lingling et al., 2007). Overall, the preliminary findings suggest that ZnO Nps can be used externally to control the spreading of bacterial infections. Using MIC test and difference in the standard growth curve, ZnO Nps at increasing concentrations decrease the growth of K. pneumoniae. It has been reported that the negative charge on the cell surface of Gram-negative bacteria was higher than on Gram-positive bacteria (Chung et al., 2004). Due to a higher negative charge on cell surface, the interaction between Gram-negative bacteria and Nps was definitely stronger than that of Gram-positive bacteria. The electrostatic interaction between the bacteria surface and Nps could decrease the growth of K. pneumoniae (Stoimenov et al., 2002). ZnO Nps block or inhibit the
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Figure 8. Bradford assay for protein leakage analysis of ZnO Nps-treated cells of K. pneumoniae. Values represent mean ± SD of triplicate experiments.
growth of K. pneumoniae in lag phase itself. In the case of untreated cells, the bacterial replication goes to stationary phase after 7 h of incubation. During the lag phase, cellular metabolism is accelerated, resulting in rapid biosynthesis of cellular macromolecules, primarily enzymes (Rolfe et al., 2012). In this study on the treatment with ZnO Nps, cells showed moderate increase in size, thereby limiting cellular metabolism and biosynthesis. This result reveals that ZnO Nps are significantly active against K. pneumoniae in the lag phase. Membrane permeability is the first step involved in the resistance of bacteria to an antibiotic. The OMPs that constitute porins play a major role in the definition of intrinsic resistance in Gram-negative bacteria that is altered under antibiotic exposure (Berry et al., 2005). OMPs of Gram-negative bacteria have diverse functions. They are directly involved in the interaction with various environments encountered by pathogenic organisms. Thus, OMPs represent important virulence factors and play essential roles in bacterial adaptation to host niches, which are usually hostile to invading pathogens. OMP and inner membrane fraction of K. pneumoniae grown in the presence of ZnO Nps showed significant difference in (i.e., decrease) protein levels ranging between 66 and 29 kDa. The cell wall of the most pathogenic bacteria is composed of surface proteins for adhesion and colonization, and components such as polysaccharides and teichoic acid that protect against host defenses and environmental conditions (Morones et al., 2005). OMP components are charged macromolecules which exhibit specific interactions with Nps and disrupt the main function. Generally, it is believed that nano-materials release ions,
which react with the thiol groups (–SH) of the proteins present on the bacterial cell surface. Such proteins protrude through the bacterial cell membrane, allowing the transport of nutrients through the cell wall. Nano-materials inactivate the proteins, decreasing the membrane permeability and eventually causing cell death (Zhang & Chen, 2009). These studies have indicated that families of unrelated hydrophobic groups are equally efficient at killing bacteria. The above mechanism may be involved in the ZnO Nps suppression of OMP expression in K. pneumonia. There is a high antibacterial effect where the ZnO Nps shows maximum effect by having direct interaction in degrading the LPS membrane. The role of LPS in adherence may be involved in non-specific physico-chemical interactions because bacterial attachment is achieved by specific surface hydrophobicity and surface charge is believed to contribute to adherence of bacteria to surface (Klotz, 1990). So in this regard, interaction that may affect attachment includes metal or other cations, polar group interaction, steric interference, and specific reaction between functional group. In our study, LPS hydrophobicity has been potentially modulated by ZnO Nps by disturbing the amino acid sequence. Maintenance of membrane potential is important for bacteria to show its virulence. According to cytoplasmic leakage analysis, the amount of nucleic acid and protein released from the cells increased along with an increasing concentration of ZnO Nps. It showed the coherence with the effect of Ag Nps against E. coli DH-5- than B. subtilis (Stoimenov et al., 2002). The sensitivity of ZnO Nps-injured cells was measured by their capacity to be lysed by SDS. Cells in which the
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Figure 9. Bacterial cell treated with ZnO nanoparticle. Supernatant of the treated cells shows the presence of nucleic acid at A260. Values represent mean ± SD of triplicate experiments.
Figure 10. Membrane stability of bacterial cell treated with ZnO Nps.
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Figure 11. Effect of ZnO Nps on catalase activity of K. pneumoniae.
Figure 12. Cytotoxicity of K. pneumoniae treated with ZnO Nps – MTT assay. Values represent mean ± SD of triplicate experiments.
Figure 13. Cytotoxicity of K. pneumoniae treated with ZnO Nps – LDH assay. Values represent mean ± SD of triplicate experiments.
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membrane deformed after treatment showed increased lasting effect and destabilize the outer membrane. It is elucidated that the ZnO Nps can disrupt the outer membrane components such as porin and LPS (Yeh et al., 2006). It is similar to the effect of Ag Nps which initially bind to the outer membrane and particles can enter into cells at higher concentration (Yeh et al., 2006). ZnO Nps decreases the synthesis of catalase, an antioxidant enzyme that protects bacteria from oxidative stress, thus exposing the bacteria to oxidative stress and prevents growth of bacteria. Gonococci pretreated with H2O2 are significantly more resistant to neutrophils than control bacteria (Keisari et al., 2001). Such resistance seems most likely to be due to increased formation of catalase. We showed that K. pneumoniae triggers a cytotoxic effect upon infection of human oesopharyngeal carcinoma cells. This process requires the presence of capsulated live bacteria during the time of infection. Our results explain the underlying mechanism behind the early findings which indicated that K. pneumoniae expressing CPS induces extensive lung tissue damage. However, just the presence of CPS is not sufficient for K. pneumoniae-induced cytotoxicity. Bacterial factor(s) together with CPS could promote cytotoxicity in the host. The lack of cytotoxicity was observed with an increasing concentration of ZnO Nps on HEp-2 cell line-infected K. pneumoniae. In conclusion, our findings allocate novel antibacterial role of ZnO NPS on K. pneumonia by rupturing the membrane of the bacterial cell wall and binding to the intracellular material. In addition, ZnO Nps are capable of blocking invasion and shown to prevent internalization by nonphagocytic cells with minimized cell death proving it as a potential antibacterial agent. These results suggest that ZnO Nps inhibit K. pneumonia by physical and chemical mechanisms regardless of drug resistance mechanism. Hence, ZnO Nps can be considered potentially as an effective bactericidal agent.
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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Jones N, Ray B, Ranjit KT, Manna AC. (2008). Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms. FEMS Microbiol Lett 279:71–6. Katsikogianni M, Missirlis YF. (2004). Concise review of mechanism of bacterial adhesion to biomaterial and of techniques used in estimating bacteria-material interaction. Eur Cells Mater 8: 37–57. Keisari Y, Wang H, Mesika A, et al. (2001). Surfactant protein D-coated Klebsiella pneumoniae stimulates cytokine production in mononuclear phagocytes. J Leukoc Biol 70:135–41. Khalil H, Villota R. (1988). Comparative study on injury and recovery of Staphylococcus aureus using microwaves and conventional heating. J Food Prot 51:181–6. Klotz S. (1990). Role of hydrophobic interactions in microbial adhesion to plastics used in medical devices. In: Doyle R, Rosenberg M, eds. Microbial Cell Surface Hydrophobicity. Washington, DC: American Society for Microbiology, 107. Lingling Z, Yunhong J, Yulon D, et al. (2007). Investigation into the antibacterial behaviour of suspensions of ZnO nanoparticles (ZnO nanofluids). J Nanopart Res 9:479–89. Mohammed Sadiq I, Chandrasekaran N, Mukherjee A. (2010). Studies on effect of TiO2 nanoparticles on growth and membrane permeability of Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. Curr Nanosci 6:381–7. Morones JR, Elechiguerra JL, Camacho A, et al. (2005). The bactericidal effect of silver nanoparticles. Nanotechnology 16:2346–53. Padmavathy N, Vijayaraghavan R. (2008). Enhanced bioactivity of ZnO nanoparticles – an antimicrobial study. Sci Technol Adv Mater 9: 035004–11. Ravishankar Rai V, Jamuna Bai A. (2011). Nanoparticles and their potential application as antimicrobials. In: Mendez-Vilas A, ed. Science against Microbial Pathogens: Communicating Current Research and Technological Advances. Formatex Research Center, 197–209. Rolfe MD, Rice CJ, Lucchini S, et al. (2012). Lag phase is a distinct growth phase that prepares bacteria for exponential growth and involves transient metal accumulation. J Bacteriol 194:686–701. Schurr JR, Young E, Byrne P, et al. (2005). Central role of toll-like receptor 4 signaling and host defense in experimental pneumonia caused by Gram-negative bacteria. Infect Immun 73:532–45. Stoimenov PK, Klinger RL, Marchin GL, Klabunde KJ. (2002). Metal oxide nanoparticles as bactericidal agents. Langmuir 18:6679–86. Yeh KM, Chang FY, Fung CP, et al. (2006). Serotype K1 capsule, rather than magA per se, is really the virulence factor in Klebsiella pneumoniae strains that cause primary pyogenic liver abscess. J Infect Dis 194:403–4. Zhang H, Chen G. (2009). Potent antibacterial activities of Ag/TiO2 nanocomposite powders synthesized by a one-pot sol-gel method. Environ Sci Technol 43:2905–10.
ORIGINAL ARTICLE
Antimicrobial activity of zinc oxide (ZnO) nanoparticle against Klebsiella pneumoniae Lanka Shalini Reddy, Mary Magar Nisha, Mary Joice, and P. N. Shilpa
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Department of Biotechnology, Sathyabama University, Chennai, Tamil Nadu, India
Abstract
Keywords
Context: Zinc oxide nanoparticles (ZnO Nps) have potential application in piezoelectric nanogenerator and in biotechnology. Objective: The antibacterial activity of ZnO Nps on Klebsiella pneumoniae (ATCC 70068) and mode of action of ZnO Nps was investigated. Methods: ZnO Nps was synthesized by a precipitation method and characterized using scanning electron microscopy (SEM) and X-ray diffraction (XRD) methods. In vitro susceptibility of K. pnumoniae of the ZnO Nps was detected using the disk diffusion method, and the minimum inhibitory concentration (MIC) value was determined using the serial dilution method. The chemical and physical interaction between the cell envelope of K. pneumonia and ZnO Nps was investigated. The effect of ZnO Nps on the cytotoxic activities of K. pneumonia was investigated using a HEp-2 cell line. Results: The MIC of ZnO Nps was found in 40 mg/ml. The standard growth curve showed that ZnO Nps of 0.75 mM inhibited K. pneumoniae after 4 h. The interaction with outer membrane protein (OMP) and lipoploysacharride (LPS) residues showed modulation in 66 kDa and 29 kDa proteins with the use of increasing concentrations of ZnO Nps. The amount of nucleic acid and protein released from the cells increased with the ZnO Nps concentration used. Importantly, the OD of the ZnO Nps-treated cells decreased within 30 min of incubation in the presence of SDS. ZnO Nps-treated K. pneumoniae were five-fold less infectious in the HEp-2 cell line at doses between 0.50 and 0.75 mM. Discussion: These results suggest the potential antibacterial use of ZnO Nps against K. pneumoniae infections.
Agar diffusion assay, growth curve, HEp-2 cells, lipoploysaccharride, metal oxide nanoparticle, minimum inhibitory concentration, outer membrane protein
Introduction The treatment of bacterial infections is increasingly complicated by the ability of bacteria to develop resistance to antimicrobial agents. There are several new strategies that have been employed to control microbial infection. Metal oxide nanoparticles, a new class of materials, are increasingly being recognized for potential use in research and healthrelated application. Recent studies have demonstrated that specially formulated metal oxides Nps possess good antibacterial potential (Azam et al., 2012) and Nps-based antimicrobial formulations could be used as an efficient bactericidal material in modern medicine (Padmavathy & Vijayaraghavan, 2008). Among several metal oxides, ZnO has recently achieved special attention regarding potential electronic application due to its unique optical, electrical, and chemical properties (Jiang et al., 2009). ZnO appears to be strongly resisted to microorganisms where as ZnO Nps exhibit strong antibacterial activities on broad-spectrum pathogenic bacteria Correspondence: Dr. P. N. Shilpa, Virtis Bio Labs, 34, Velautham Gounder Complex, Dharman Nagar, Ammapalayam Main Road, Salem 636 005, Tamil Nadu, India. Tel: +91 9597926827. E-mail: [email protected]
History Received 9 March 2013 Revised 28 December 2013 Accepted 6 February 2014 Published online 15 July 2014
such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, E. coli O157:H7, Salmonella enteritidis, Salmonella typhimurium, Pseudomonas fluorescens, and Listeria monocytogenes (Adams et al., 2006; Huang et al., 2008; Jones et al., 2008; Ravishankar Rai & Jamuna Bai, 2011). ZnO nanoparticles (12 nm) inhibited the growth of E. coli by disintegrating the cell membrane and increasing the membrane permeability (Huh & Kwon, 2011). Recent lines of evidence suggest that the synthesis of hydrogen peroxide (Ravishankar Rai & Jamuna Bai, 2011) and eventual penetration of cell envelope and disorganization of bacterial membrane following contact with ZnO Nps are the two potential mechanisms linked to bacterial inhibition (Brayner et al., 2006; Huang et al., 2008). However, very few detailed studies were conducted on the relationship between the ZnO Np and the antibacterial activity. Klebsiella pneumoniae is an important pathogen frequently implicated in respiratory and urinary tract infections (UTI) of hospitalized patients. It is a part of the respiratory and intestinal microbiota of humans and is isolated from the oropharyngeal cavity at a frequency of 1–6% (Mohammed et al., 2010). It is also strongly associated with infections namely septicemias, and iatrogenic infections, since the
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hospital environment is associated with long-staying hospitalized patients and/or those who have been in the intensive care unit (ICU), under oropharyngeal intubation. Between half and three-quarters of all bacteremias caused by K. pneumoniae are nosocomial and are resistant to ampicillin and carbenicillin, albeit being frequently susceptible to cephalosporins, cotrimoxazole, aminoglycosides, and imipenem (Archana & Harsha, 2011). The growth inhibition of K. pneumoniae has been tested using several inorganic nano-metals by others (Ravishankar Rai & Jamuna Bai, 2011). Nonetheless, ZnO showed greater bacterial growth inhibition as a result of potential intracellular incorporation of Nps (Jones et al., 2008). Notwithstanding the effectiveness of ZnO as an antimicrobial agent, questions remain as to whether bacteria will develop resistance to ZnO (similar to that seen with modern antibiotics). Numerous models have been suggested including deactivation of certain bacterial enzymes, disruption of gene replication, and limitation of cellular membrane function (Mohammed et al., 2010). However, the true mechanism for ZnO antibacterial action remains uncertain. The cell wall of most pathogenic bacteria is composed of complex surface proteins for adhesion and colonization, and components such as polysaccharides and teichoic acid that protect against host defenses and environmental conditions (Katsikogianni & Missirlis, 2004). These components are charged macromolecules, and, therefore, specific interactions to disrupt their main functions and location may be triggered by introducing specific groups on the surface of the microorganism. Hence, we set out to investigate the chemical and physical interaction between the cell envelope of K. pneumonia and ZnO Nps. Further, investigations were also undertaken to investigate the effect of ZnO Nps on the cytotoxic activities of K. pneumonia using a HEp-2 cell line.
Materials and methods Bacteria Standard strain of Klebsiella pneumoniae (ATCC 70068) was collected from Global Hospital, Chennai, India. For routine use, the cultures were maintained on nutrient agar (NA) (Hi-media, Mumbai, India) plates/slants. Glycerol stocks were prepared and placed at 20 C for long-term storage. Cell line Human esopharyngeal carcinoma cell line (HEp-2) was purchased from KIPM (King Institute of Preventive Medicine, Chennai-32, India), and grown in minimal essential media supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 50 IU/ml penicillin, and 50 mg/ml streptomycin, incubated at 37 C under humidified atmosphere with 5% CO2. ZnO nanoparticles ZnO Nps was synthesized by a standard method (Padmavathy & Vijayaraghavan, 2008). This was also used to investigate the effect of ZnO Nps on the bacterial samples held at varying concentrations 0.25, 0.50, and 0.75 mM by performing a series of other investigations described by Archana and Harsha (2011).
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Characterization of ZnO Nps Phase purity and grain size were determined by XRD analyses recorded on a Siefert X-ray diffractometer (Siefert, Germany) ˚ ) at 60 keV over the using CuKa radiation (l ¼ 1.54016 A range of 20–70 . The average grain size was determined by X-ray line broadening using the Scherrer equation after incorporating corrections. The morphology and size of the particle were determined using a SEM (Hitachi S-4500, Hitachi, Shiga, Japan). MIC and growth curve Test organism K. pneumoniae were grown separately in a 50 ml sterilized nutrient broth medium and kept in a shaker incubator at 37 C for 14 h (overnight incubation). On the subsequent day, test organism cultures were transferred at the rate of 1% in 100 ml nutrient broth. MIC assay of ZnO Nps The initial concentration (8 mg/ml) was diluted using double fold serial dilution by transferring 5 ml of the sterile ZnO Nps (stock solution) into 5 ml of sterile nutrient broth to obtain 4 mg/ml concentration. The above process was repeated several times to obtain other dilutions. To different concentrations of ZnO Nps, 0.1 ml of the standardized bacterial cell suspension was added and incubated at 37 C for 24 h. The growth of the inoculums in the broth is indicated by turbidity or cloudiness of the broth and the lowest concentration of the ZnO Nps, which inhibited the growth of the test organism, were taken as the minimum inhibitory concentration (MIC). Growth curve Various concentrations of ZnO Nps (0.25, 0.50, and 0.75 mM) were carefully placed into each flask, leaving one as a control to track the normal growth of the microbial cells without nanoparticles. Experiments were performed using both a negative control (flask containing cells plus media) and a positive control (flask containing nanoparticles plus media). The flasks were shaken at 180 rpm at 37 C in a shaker incubator. Optical density measurements from each flask were taken every 1 h to record the growth of the microbes in a spectrophotometer set at 600 nm. The growth rate of microbial cells interacting with the nanoparticles was determined from a plot of the log of the optical density versus time. Protein fractionation Outer membrane proteins (OMPs) were isolated from overnight cultures of K. pneumoniae that have been incubated with 0.25, 0.50, and 0.75 mM of ZnO Nps and centrifuged for 5 min at 7000 g. Pellets were washed once with 20 mM Tris, 10 mM EDTA, pH 8 (TE), and resuspended in the same buffer. Bacteria were disrupted by sonication for 1 min, followed by 2 min blank, and an additional 1 min sonication. Samples were centrifuged for 5 min at 7000 g to remove debris, and the resulting supernatant was centrifuged for 1 h at 60 000 g at 4 C. The clear supernatant was retained as cytosolic fraction. The pellet was resuspended in TE buffer and the protein concentration was estimated. Protein concentration was adjusted to 5 mg/ml and solubilizes with sodium
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lauryl sarcosinate 1% w/v (final concentration) at 4 C for 1 h. Samples were centrifuged again for 1 h at 60 000 g at 4 C and the supernatant was taken for analysis of inner membrane fractions. The pellet containing OMP was resuspended in 1% SDS and boiled for 10 min. The resulting sample was run on an SDS-PAGE unit (Genei, Bangalore, India) by adding 5 ml of loading dye to the samples. Following electrophoresis, the gel was stained in commassie brilliant blue (CBB) for overnight and was destained to view the protein bands using a transilluminator (Bharathi et al., 2008).
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LPS extraction LPS was extracted by a standard method (Galanoso et al., 1969). Briefly, the overnight culture of bacterial suspension (108 CFU units/ml) incubated with 0.25, 0.50, and 0.75 mM ZnO Nps was centrifuged at 10 000 g. The pellet was washed with PBS (pH 7.2) and resuspended in water. Subsequently, equal volumes of hot (65–70 C) phenol was added and stirred vigorously. The suspension was brought to room temperature and centrifuged at 8500 g. The supernatant was transferred to a conical centrifuge tube. Phenol was re-extracted from the preparation and aqueous solution was pooled. Sodium acetate (0.5 M) and 10 volume of 95% ethanol were added, and placed overnight at 20 C in order to precipitate the LPS. Following a brief centrifugation at 2000 g at 4 C, the final pellet was suspended in d.H2O and the final precipitate was used in SDS-PAGE. Protein leakage analysis Protein leakage analysis was performed using a standard Bradford assay (Bradford, 1967). Briefly, K. pneumoniae cells were treated with 0.25, 0.50, and 0.75 mM ZnO Nps in nutrient broth (pH 7.4) for 1 h and 5 h at 37 C. Supernatant was collected after centrifugation (6000 rpm) for 15 min. For each sample, 200 ml of the supernatant was mixed in 800 ml of the Bradford reagent. The optical density (595 nm) was measured after 10 min of incubation in the dark. BSA was used as a standard protein. Membrane stability of K. pneumoniae The effect of ZnO Nps on the outer membrane of the bacteria was checked by SDS treatment of pre-treated cells (with ZnO Nps) of K. pneumoniae. Active culture of K. pneumoniae was mixed in PBS-buffer containing 0.25, 0.50, and 0.75 mM ZnO Nps and incubated for 30 min. Cells were centrifuged and the pellet was mixed in the same volume of PBS. SDS (0.15%) was mixed in ZnO Nps-treated cells. OD was measured every 2 min. Nucleic acid analysis K. pneumoniae cells were treated with 0.25, 0.50, and 0.75 mM ZnO Nps in nutrient broth (pH 7.4) at 37 C. Supernatant was collected after centrifugation (6000 rpm) for 15 min. The amount of nucleic acid released in 0.25, 0.50, and 0.75 mM ZnO Nps-treated cells was measured at 260 nm using a UV–VIS spectrophotometer. For preliminary identification of nucleic acid leakage, ninhydrin test showed a positive result. The experiment was repeated for minimizing error (Khalil & Villota, 1988).
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Isolation of catalase Cell extracts of bacteria incubated with 0.25, 0.50, and 0.75 mM were collected at the mid-logarithmic or stationary phase by centrifugation at 10 000 g for 5 min at 4 C. Pellets were washed once, resuspended in 200 ml of electrophoresis buffer, and sonicated twice in an ice water bath for 30 s with a sonicator. The sonicate was then centrifuged at 15 000 g for 20 min at 4 C and native PAGE (10%) was run. The gel was soaked in d.H2O to remove trace of running buffer. The gel was soaked in 200 ml of 3 mM solution of H2O2 for 10 min, rinsed in d.H2O and incubated in 1% potassium ferric cyanide for 10 min in the dark. Cytotoxicity assay HEp-2 cells were grown in MEM supplemented with 5% FCS, 2 mM L-glutamine, and 50 IU/ml penicillin and 50 mg/ml streptomycin for 3–4 d in a CO2 incubator. HEp-2 cells were grown to confluence in 96-well plates. The confluent monolayer was loaded with different concentrations of ZnO Nps and equal multiplicity of infection (MOI) (50:1) of K. pneumoniae. The percentage of cytotoxicity was assessed by MTT as well as lactate dehydrogenase (LDH) assay (Das Niranjali, 2008).
Results The ZnO Nps was synthesized by the precipitation and surface modification methods. The XRD patterns of ZnO samples prepared by the two methods are shown in Figure 1. Figure 1(a) shows the peak broadening of the XRD pattern for the precipitation method. With increasing annealing temperature, the crystalline nature of the sample appears to improve and the grain size increased. The broadened peaks in the XRD pattern obtained by surface modification (Figure 1b) indicate the formation of ZnO nanocrystals with small crystallites. The crystallite size obtained by the two synthesis methods was estimated by the Scherrer equation and found to be in the range of 20–40 nm. Characterization of ZnO Nps was confirmed by SEM analysis where it shows the variations in particle size of 159 nm in the precipitation method and 88.7 nm of reduced size by the surface modification method. The particle interaction and aggregation are observed in ZnO Np obtained by the precipitation method. The presence of capping molecules appears to affect the kinetics of nucleation and accumulation in such a way that the rate of growth of large particle decreases while that of small particles remains the same. This resulted in a narrowing of the size change of the particles after the addition of a capping molecule. The reduction in size was observed by surface modification method than compared with the precipitation method (Figure 2a and b). The representative results of the bacterial count for different concentrations for ZnO Nps for K. pneumonia is shown in Figure 3. By doing serial dilution, ZnO Nps at different concentrations showed a dramatic decrease in the number of colonies formed. Serially with decreasing concentrations of Nps, there will be an increase in the bacterial growth. Thus, with increased Nps concentration, greater antibacterial effect was observed. Figure 4 show the effect of
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Figure 1. (a) XRD pattern of ZnO Nps formed by precipitation method. (b) XRD pattern of ZnO Nps after surface modification.
Figure 2. (a) Characterization of ZnO Nps synthesized by the precipitation method using SEM analysis. (b) Modification of ZnO Nps by the surface modification method using SEM analysis.
ZnO NPS on the growth of K. pneumoniae where timedependent changes in bacterial growth are monitored by measuring OD at 595 nm. The OD at 595 nm is due to the scattering of light by the bacterial cells. It is a function of bacterial cell density and thus correlates with the growth of
the colonies. It is clear that ZnO Nps at increasing concentration decreases the growth of K. pneumoniae. At concentration 0.75 mM, ZnO NPs showed potential inhibition after 4 h as compared with the standard growth curve of K. pneumoniae.
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Figure 3. Minimum inhibitory concentration test of ZnO Nps against K. pneumoniae. Values represent mean ± SD of triplicate experiments.
Figure 4. Growth curve of K. pneumoniae treated with ZnO Nps.
K. pneumoniae was grown overnight at 37 C in medium with and without varied concentrations of ZnO Nps (0.25, 0.50, and 0.75 mM). The proteins were fractioned and subjected to SDS-PAGE. Figures 5 and 6 show the effect of the different concentrations of ZnO Nps on the K. pneumoniae OMP and inner membrane fractions. In the presence of ZnO Nps, the outer membrane as well as the inner membrane fractions showed significant inhibition at higher levels of 66 kDa and lower levels of 29 kDa relative to K. pneumonia grown in the absence of ZnO Nps. In addition, there were some other minor differences between protein
profiles of the bacteria grown in the presence and absence of ZnO Nps. There was a significant diminishing of protein bands (Figure 7) after silver staining. The ZnO Nps showed its maximum effect by direct interaction with the LPS, thereby reducing the virulence of the pathogen. The LPS was degraded in a dose-dependent manner. The amount of nucleic acid released into the suspension was analyzed by the measuring absorbance at 260 nm. The amount of protein released into suspension of treated cells was estimated by the Bradford assay. The amount of protein (Figure 8) and nucleic
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Figure 5. Effect of ZnO Nps on the outer membrane protein of K. pneumoniae. Lane 1: control, Lane 2: 0.25 mM ZnO Nps, Lane 3: 0.5 mM ZnO Nps, Lane 4: 0.75 mM ZnO Nps.
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suspension, no significant decrease in OD was observed after 30 min, either in the presence or absence of ZnO Nps. The result validates the fact that most of the cells inactivated by ZnO Nps remain unlysed in cell suspension in the absence of SDS, and these are highly sensitive to lysis by SDS. It is clear from Figure 10 that the treatment of Nps destabilizes the outer membrane. Inhibition of catalase activity was observed with an increase in the concentration of ZnO Nps showing the iota of oxidative stress induced against the bacteria (Figure 11). In the present study, K. pneumonia-induced cytotoxicity and the protection, if any rendered by ZnO Nps, were determined by two methods, MTT assay and LDH leakage. We observed that the cytotoxicity was more of the same MOI in LDH assay than MTT assay. LDH assay determines the total enzyme leakage by the defoliated as well as the adhered cells in a tissue culture plate, whereas MTT assay measures only the adhered viable cells. In MTT (Figure 12) and LDH assays (Figure 13) the maximum cytotoxicity was observed in cells infected with wild bacteria. The cytotoxicity was almost a half and five-fold less for cells infected with ZnO Npstreated bacteria at a dose of 0.50 mM and 0.75 mM, respectively.
Discussion
Figure 6. Effect of ZnO Nps on the inner membrane protein of K. pneumoniae. Lane 1: control, Lane 2: 0.25 mM ZnO Nps, Lane 3: 0.5 mM ZnO Nps, Lane 4: 0.75 mM ZnO Nps.
Figure 7. Effect of ZnO Nps on lipoploysacharride of K. pneumoniae. Lane 1: control, Lane 2: 0.25 mM ZnO Nps, Lane 3: 0.5 mM ZnO Nps, Lane 4: 0.75 mM ZnO Nps.
acid (Figure 9) released from the cells was directly proportional to the concentration of ZnO Nps used. These results indicate that most of the Nps-treated cells were ghost cells, which released intracellular material into the cell suspension. The OD of ZnO Nps-treated cells reduced dramatically within 30 min of incubation in the presence of SDS; but it did not decrease significantly in the absence of SDS. In untreated cell
The indiscriminate use of antibiotics in the modern society has led to evolution of novel pathogenic antibiotic-resistant bacterial strains. K. pneumoniae is an important human pathogen that has been associated in recent decades with nosocomial outbreaks. Following the use of extendedspectrum cephalosporins, extended-spectrum b-lactamase (ESBL)-producing K. pneumoniae has become an increasingly serious problem worldwide (Hickling et al., 2004; Schurr et al., 2005). Recent evidence suggests that an antibiotic formulation in the form of Nps could be a highly effective bactericidal agent (Huh & Kwon, 2011). This would provide a solution to the increasingly urgent problem of antibiotic resistance, reducing the risk from infections and related complications, which take a heavy toll on vulnerable hospital patients. Outbreaks of antibiotic-resistance bacteria fuel incentives to develop newer effective bactericidal agent (Mohammed et al., 2010). The experimental results so far suggest the possible antimicrobial mechanisms of suspensions of ZnO particles: physical mechanism by attaching onto the bacterial cell walls directly, biological mechanism by interacting with the cell membrane components and chemical mechanism by producing active species (Lingling et al., 2007). Overall, the preliminary findings suggest that ZnO Nps can be used externally to control the spreading of bacterial infections. Using MIC test and difference in the standard growth curve, ZnO Nps at increasing concentrations decrease the growth of K. pneumoniae. It has been reported that the negative charge on the cell surface of Gram-negative bacteria was higher than on Gram-positive bacteria (Chung et al., 2004). Due to a higher negative charge on cell surface, the interaction between Gram-negative bacteria and Nps was definitely stronger than that of Gram-positive bacteria. The electrostatic interaction between the bacteria surface and Nps could decrease the growth of K. pneumoniae (Stoimenov et al., 2002). ZnO Nps block or inhibit the
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Figure 8. Bradford assay for protein leakage analysis of ZnO Nps-treated cells of K. pneumoniae. Values represent mean ± SD of triplicate experiments.
growth of K. pneumoniae in lag phase itself. In the case of untreated cells, the bacterial replication goes to stationary phase after 7 h of incubation. During the lag phase, cellular metabolism is accelerated, resulting in rapid biosynthesis of cellular macromolecules, primarily enzymes (Rolfe et al., 2012). In this study on the treatment with ZnO Nps, cells showed moderate increase in size, thereby limiting cellular metabolism and biosynthesis. This result reveals that ZnO Nps are significantly active against K. pneumoniae in the lag phase. Membrane permeability is the first step involved in the resistance of bacteria to an antibiotic. The OMPs that constitute porins play a major role in the definition of intrinsic resistance in Gram-negative bacteria that is altered under antibiotic exposure (Berry et al., 2005). OMPs of Gram-negative bacteria have diverse functions. They are directly involved in the interaction with various environments encountered by pathogenic organisms. Thus, OMPs represent important virulence factors and play essential roles in bacterial adaptation to host niches, which are usually hostile to invading pathogens. OMP and inner membrane fraction of K. pneumoniae grown in the presence of ZnO Nps showed significant difference in (i.e., decrease) protein levels ranging between 66 and 29 kDa. The cell wall of the most pathogenic bacteria is composed of surface proteins for adhesion and colonization, and components such as polysaccharides and teichoic acid that protect against host defenses and environmental conditions (Morones et al., 2005). OMP components are charged macromolecules which exhibit specific interactions with Nps and disrupt the main function. Generally, it is believed that nano-materials release ions,
which react with the thiol groups (–SH) of the proteins present on the bacterial cell surface. Such proteins protrude through the bacterial cell membrane, allowing the transport of nutrients through the cell wall. Nano-materials inactivate the proteins, decreasing the membrane permeability and eventually causing cell death (Zhang & Chen, 2009). These studies have indicated that families of unrelated hydrophobic groups are equally efficient at killing bacteria. The above mechanism may be involved in the ZnO Nps suppression of OMP expression in K. pneumonia. There is a high antibacterial effect where the ZnO Nps shows maximum effect by having direct interaction in degrading the LPS membrane. The role of LPS in adherence may be involved in non-specific physico-chemical interactions because bacterial attachment is achieved by specific surface hydrophobicity and surface charge is believed to contribute to adherence of bacteria to surface (Klotz, 1990). So in this regard, interaction that may affect attachment includes metal or other cations, polar group interaction, steric interference, and specific reaction between functional group. In our study, LPS hydrophobicity has been potentially modulated by ZnO Nps by disturbing the amino acid sequence. Maintenance of membrane potential is important for bacteria to show its virulence. According to cytoplasmic leakage analysis, the amount of nucleic acid and protein released from the cells increased along with an increasing concentration of ZnO Nps. It showed the coherence with the effect of Ag Nps against E. coli DH-5- than B. subtilis (Stoimenov et al., 2002). The sensitivity of ZnO Nps-injured cells was measured by their capacity to be lysed by SDS. Cells in which the
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Figure 9. Bacterial cell treated with ZnO nanoparticle. Supernatant of the treated cells shows the presence of nucleic acid at A260. Values represent mean ± SD of triplicate experiments.
Figure 10. Membrane stability of bacterial cell treated with ZnO Nps.
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Figure 11. Effect of ZnO Nps on catalase activity of K. pneumoniae.
Figure 12. Cytotoxicity of K. pneumoniae treated with ZnO Nps – MTT assay. Values represent mean ± SD of triplicate experiments.
Figure 13. Cytotoxicity of K. pneumoniae treated with ZnO Nps – LDH assay. Values represent mean ± SD of triplicate experiments.
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membrane deformed after treatment showed increased lasting effect and destabilize the outer membrane. It is elucidated that the ZnO Nps can disrupt the outer membrane components such as porin and LPS (Yeh et al., 2006). It is similar to the effect of Ag Nps which initially bind to the outer membrane and particles can enter into cells at higher concentration (Yeh et al., 2006). ZnO Nps decreases the synthesis of catalase, an antioxidant enzyme that protects bacteria from oxidative stress, thus exposing the bacteria to oxidative stress and prevents growth of bacteria. Gonococci pretreated with H2O2 are significantly more resistant to neutrophils than control bacteria (Keisari et al., 2001). Such resistance seems most likely to be due to increased formation of catalase. We showed that K. pneumoniae triggers a cytotoxic effect upon infection of human oesopharyngeal carcinoma cells. This process requires the presence of capsulated live bacteria during the time of infection. Our results explain the underlying mechanism behind the early findings which indicated that K. pneumoniae expressing CPS induces extensive lung tissue damage. However, just the presence of CPS is not sufficient for K. pneumoniae-induced cytotoxicity. Bacterial factor(s) together with CPS could promote cytotoxicity in the host. The lack of cytotoxicity was observed with an increasing concentration of ZnO Nps on HEp-2 cell line-infected K. pneumoniae. In conclusion, our findings allocate novel antibacterial role of ZnO NPS on K. pneumonia by rupturing the membrane of the bacterial cell wall and binding to the intracellular material. In addition, ZnO Nps are capable of blocking invasion and shown to prevent internalization by nonphagocytic cells with minimized cell death proving it as a potential antibacterial agent. These results suggest that ZnO Nps inhibit K. pneumonia by physical and chemical mechanisms regardless of drug resistance mechanism. Hence, ZnO Nps can be considered potentially as an effective bactericidal agent.
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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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