Synergistic activity of doped zinc oxide nanoparticles with antibiotics: ciprofloxacin, ampicillin, fluconazole and amphotericin B against pathogenic microorganisms

Combination therapy of antibiotics and nanoparticles can be used against multi drug resistant microorganisms. Nanoparticles (NPs) have been reported to show antimicrobial activity. The antimicrobial activities of doped ZnO nanoparticles (ZnO NPs) were studied against fungi, gram-positive and gram-negative bacteria using the standard microdilution method. The interaction between the nanoparticle and the antibiotic was estimated by calculating the fractional inhibitory concentration (FIC index) of the combination through checkerboard assay. Experimental results demonstrated that 10% doped zinc oxide nanoparticles (ZnO NPs) exhibited the maximum antimicrobial effect in contrast with that of the 1% loading and pure ZnO nanoparticles. The enhancement in antimicrobial effect was seen when combined with antibiotic. Synergistic and additive effects were found. No antagonistic effect was found. More synergistic effect was observed when combined with ciprofloxacin than ampicillin. Fungus showed only additive effect. The results are quite in terms with MIC clearly depicting that high doping agent is most suitable for combined therapy. 100% synergistic interaction was observed in higher doping with both ciprofloxacin and ampicillin. This study provides a preliminary report of the synergistic activity of nanoparticles with antibiotics against different pathogenic strains. This provides groundwork for further studies on the combination therapy of nanoparticles with antibiotics.


INTRODUCTION
The increasing and indiscriminate usage of antibiotics and poor patient compliance has led to the development of bacterial immunity to antibiotics. Around the world, as much as 60% of hospital-acquired infections are caused by drugresistant microorganisms (Edmond et al. 1999). The organism has successfully developed numerous strategies for resisting the action of practically all antibiotics (Kuroda et al. 2001). Currently used antifungal agents also have toxic sideeffects, may interact with other drugs, and become ineffective as a consequence of the rapid growth of fungal resistance (Hammer et al. 1998, Shahi et al. 1999. Moreover, the therapeutic response may be slow, and thus inappropriate for treatment of patients with severe or rapidly progressive mycoses. This resistance to antimicrobial agents NEHA SHArMA, SAvITA JANDAIK and SANJEEv KUMAr has resulted in morbidity and mortality from treatment failures, and increased health care costs. Although determining the precise public health risk and estimating the gain in prices is not a simple task, there is the slight question that the emerging antibiotic resistance is a serious worldwide problem. Likewise, the development of vaccines and new antimicrobial agents has not kept pace with resistance; therefore, the search for other methods of therapy, such as synergistic combinations, is necessary. Combination therapy is applied with the intention of expanding the antimicrobial spectrum, minimizing toxicity, preventing the emergence of resistant mutants during therapy and obtaining synergistic antimicrobial activity (Eliopoulos and Moellering Jr 1991). The increased clinical response to combination therapy is explained to be due to synergism between the antibiotics used. Synergism of a combination of antibiotics can be stated as fractional inhibitory concentration indices (FICi) derived from a checkerboard titration. Synergism has been defined as a phenomenon in which two different compounds are combined to enhance their individual activity. If the combination results in worsening effect, it is called antagonism. An effect which is less than synergistic but not antagonistic is termed as additive or indifference (rani et al. 2009).
Nanotechnology represents a modern and innovative approach to develop new formulations based upon metallic nanoparticles with antimicrobial properties. A probe of the interactions of antibiotics with silver (Ag) nanoparticles is the most common among studies dedicated to the testing of combined action of nanoparticles with antibiotics. Few studies have found that the efficacy of antimicrobial agents can be improved by combining them with nanoparticles against different pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, etc. recently, some metal nano particles have been valued for increasing the antibacterial actions of different antibiotics. Therapeutic roles for zinc in different diseases have been established in recent years. Zinc oxide has a very good potential to get into the clinic (Shopsin et al. 1999). Studies have revealed improved activity of nano ZnO when used in combination with cephalosporins, beta lactums and amino glycosides against different pathogenic microorganisms (Gaddad et al. 2010, Solomon et al. 2007.
The purposes of the present investigations were to evaluate the antimicrobial activity of the nanoparticles and compare them with the effect of antibiotics on the increasing resistance of different pathogenic microbes used; and to evaluate the interaction of the nanoparticles and antibiotics on these lines.

materiaLS aNd methOdS
Microrganisms: In the present study, six bacterial and two fungal isolates were procured from GianSagar Medical College, rajpura, Punjab. The isolates were identified by conventional methods. Standard cultures (s) of bacteria and fungi were procured from Institute of Microbial Technology (IMTECH), Chandigarh. Following bacterial and fungal isolates were chosen: Minimum Inhibitory Concentration range for undoped ZnO was 0.16-0.43 mg/ml in case of bacteria. The most effective value of MIC was obtained against S. typhi (s and c) and E. coli (s) at 0.16±0.01 mg/ml (s and c) and 0.16±0.02 mg/ ml concentrations respectively. MIC observed for T. mentegrophytes (s) and C. neoformens (c) was 0.33±0.14 mg/ml and 26.6±14.4 mg/ml respectively.
Excellent minimum inhibitory concentration of 0.09 mg/ml was obtained with 10% iron (Fe) doping for B. subtilis (s) followed by 0.11 mg/ml for E. coli (s) and 0.12 mg/ml for S. aureus (c) at the same doping concentration. In case of 1% Fe doping, of all the pathogenic microbes used S. typhi (s and c) showed best results at 0.13 mg/ml minimum inhibitory concentration. MIC values for both the fungal strains, i.e. T. mentegrophytes (s) and C. neoformens (c) were 0.25±0.21 mg/ml and 11.5±11.8 in case of 10% Fe doping and 0.33±0.14 mg/ml and 13.5±10.9 mg/ml in case of 1% Fe doping respectively.
An admirable minimum inhibitory concentration of 0.004±0 mg/ml against B. subtilis (s) was obtained with 10% copper (Cu) doping which was   Interaction of nanoparticles with the antimicrobial agents was assessed by checkerboard as-say as summed up in tables II-Iv. Synergistic and additive effects were found, no antagonistic effect was seen. The results are quite in terms with MIC clearly depicting that high doping agent is most effective antimicrobial agents. Though not much varied, but more synergistic effect was observed in when doped nanoparticles were combined with ciprofloxacin as compared to ampicillin. Undoped ZnO nanoparticles showed a synergistic effect when combined with ciprofloxacin against K. pneumoniae only. Additive effect was only observed for undoped ZnO nanoparticles when combined with ampicillin. Ciprofloxacin exhibited 100% synergism (12/12 strains) for 10% doped nanoparticles i.e. 10% Fe, 10% Mn, 10% Cu and 10% Co doped ZnO nanoparticles. The least synergistic effect

DISCUSSION
The issue of resistance to antibiotics and its diffusion, however, are major health troubles, leading to treatment drawbacks for a large number of drugs (Braga et al. 2005, Schito 2006. Drug resistance enforces high dose administration of antibiotics, often generating intolerable toxicity, development of new antibiotics, and requests for significant economic, labor, and time investments. It has been assumed that if present trends continue, antibiotic failure will claim 10 million lives per year by 2050 (O'Neill 2014). Therefore, there has been increasing interest in the role of inhibitors of antibiotic resistance for combination therapy (Gibbons 2005, Wright 2005). Nanotechnology represents a modern and forward-looking approach to develop new formulations based upon metallic nanoparticles with antimicrobial properties. Therapeutic roles for zinc in different diseases have been demonstrated in recent years.
With the goal of developing a new highly active antimicrobial therapeutic combination of nanoparticles and synthetic antimicrobial agents, we began by comparing the antimicrobial properties of nine nanoparticles against different pathogenic bacterial and fungal strains. Higher doped nanoparticles (10%) revealed better antimicrobial property than the lesser doped (1%) and undoped. The MIC against test strains shows that nanoparticles have a less significant effect on growth of gram-positive bacteria than on gram-negative bacteria. This is due to the structural difference in cell wall composition of gram-positive and gramnegative bacteria. The gram-negative bacteria have a layer of lipopolysaccharides at the exterior, followed underneath by a thin (∼7-8 nm) layer of peptidoglycan (Madigan and Martinko 2005). Although the lipopolysaccharides are composed of covalently linked lipids and polysaccharides, there is a lack of strength and rigidity. The negative charges on lipopolysaccharides are attracted toward the weak positive charge available on the doped and undoped ZnO nanoparticles (Sui et al. 2006). On the other hand, the cell wall in gram-positive bacteria are principally composed of a thick layer (∼20-80 nm) of peptidoglycan consisting of linear polysaccharide chains cross-linked by short peptides to form a three-dimensional rigid structure (Baron 1996). The rigidity and extended cross-linking not only endow the cell walls with fewer anchoring sites for the doped and undoped ZnO nanoparticles but also make them difficult to penetrate.
regards to mycoses, nanoparticles can be considered as a potential antifungal agent. Our results showed better antifungal activity of ZnO nanoparticles against T. mentagrophytes, as compared to earlier studies of Sawai and Yoshikawa (2004). The antifungal effect of doped and undoped ZnO nanoparticles particularly the doped has received only marginal attention and just a few studies on this topic has been published. Our study suggests NEHA SHArMA, SAvITA JANDAIK and SANJEEv KUMAr the better antifungal activity of doped nanoparticles than pure nanoparticles. Thereby, further supporting the fact that doping increases antimicrobial activity.
There is a significant difference in the MIC values of undoped ZnO nanoparticles and doped ZnO nanoparticles. This indicates the fact that dopant was interfering with the active principle as the MIC values are greater in doped ZnO nanoparticles as compared to undoped ZnO nanoparticles for different groups of bacteria and fungi. It has been found from some previous studies that doping may increase the antimicrobial effect (rekha et al. 2010). Dopant impurities like Cu 2+, Mn 2+ , Co 2+ , Ni 2+ , rare earth and transition elements, brings about significant changes in the physical, chemical and biological properties of host material on doping depending on the type of dopant and its concentration (Peng et al. 2006, Zhang et al. 2006. The increase in antibacterial and photocatalytic activity with increase in dopants concentration is attributed to the generation of reactive oxygen species (rOS).
For many years, antimicrobial drugs have been used to inhibit or kill bacteria and other microbes. However, microbial resistance to these drugs has developed on a very large scale over time, greatly reducing their effectiveness, and is an ever growing problem (Hajipour et al. 2012). One of the most promising strategies for overcoming microbial resistance is the use of nanoparticles. Development of resistance to these nanoparticles is, again, unlikely (Friedman et al. 2013), possibly because it would require multiple simultaneous gene mutations in the same microbial cell. Therefore, the interaction of nanoparticles with commonly used antimicrobials was studied by checkerboard method. The checkerboard test measures the inhibitory concentration. Here also results revealed better activity of doped nanoparticles than the undoped. Further strengthening the fact that doping increase the antimicrobial activity. recent studies have shown that combining nanoparticles with antibiotics not only reduces the toxicity of both the agents towards human cells by decreasing their requirement or high dosages but also enhances their antimicrobial properties. Combining antibiotics with nanoparticles also restores their ability to destroy microbes that have acquired resistance to them. Furthermore, nanoparticles tagged with antibiotics have been shown to increase the concentration of the antibiotics at the site of bacterium-antibiotic interaction, and facilitate in the binding of antibiotics to microorganisms (Allahverdiyev et al. 2011). Our results are in terms with the study conducted by Banoee et al. (2010) showing better activity of ciprofloxacin nanoparticle combination as compared to ampicillin nanoparticle combination. They reported 27 and 22% increase in inhibition zone areas was observed against S. aureus and E. coli, respectively when ZnO nanoparticles combine with ciprofloxacin. However, our study is contradictory to the study conducted by Gaddad et al. (2010) who reported good activity of ampicillin and moderate activity of ciprofloxacin against S. aureus. Increased activity of ciprofloxacin in presence of doped ZnO nanoparticles can be attributed to inhibition of ciprofloxacin efflux from the cell due to interference of the doped ZnO nanoparticles with pumping NorA protein, activation of ciprofloxacin uptake by influencing activity of membrane Omf protein, and by binding reaction between ciprofloxacin and ZnO nanoparticles stabilizing the ciprofloxacindoped ZnO nanoparticle complex. This is done by the presence of fluore group and carboxyl group on the ciprofloxacin. The fluore group interacts with the chelating Zn atom stabilizing the ciprofloxacinnanoparticle combination whereas carboxyl group serves as an obvious target for chelation by metal ions (Banoee et al. 2010). Moreover, presence of doping ion on ZnO nanoparticle surface accelerates generation of reactive oxygen species (rOS) due to coupling or synergistic effects of Mn, Co, Cu and Fe loading. Further with an increase in concentration of doping synergistic activity increased as interaction between oxygen and dehydrogenase enzyme increases which increases the activity (Sikong et al. 2010). Minimal enhanced or unchanged antimicrobial activity of the other tested antibiotics against bacteria and funguscan be elucidated either by formation of weak hydrogen bonds with hydroxylated doped and undoped ZnO nanoparticles or by lack of sufficient targets for interaction (Banoee et al. 2010).