Antonie van Leeuwenhoek DOI 10.1007/s10482-015-0449-8
ORIGINAL PAPER
Evidence for bactericidal activities of lipidic nanoemulsions against Pseudomonas aeruginosa Neeru Singh . Saurabh Manaswita Verma . Sandeep Kumar Singh . Priya Ranjan Prasad Verma
Received: 18 November 2014 / Accepted: 4 April 2015 Ó Springer International Publishing Switzerland 2015
Abstract Pseudomonas aeruginosa has been implicated in a broad range of infections and shown to acquire rapid resistance to anti-microbial agents. In the present, study we have used particular amalgamation of specific lipids that hold innate antibacterial activities, which can be transformed into cationized and non-cationized nanoemulsions. The anti-Pseudomonas activities were then elucidated by transmission/scanning electron microscopy, and atomic force microscopy. The microscopic studies revealed the cell lysis due to the formation of blebs, exudation of essential cellular contents and loss of characteristics contour of the cells. The microscopic studies were then corroborated by zone of inhibition, cytoplasmic release studies, time dependent killing and MIC determination. Conclusively, it can be inferred that the delivery issues of antibiotics could be reassessed by using certain excipients that possess inherent
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0449-8) contains supplementary material, which is available to authorized users. N. Singh (&) Division of Biomedical Lab Technology, University Polytechnic, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India e-mail: [email protected] S. M. Verma S. K. Singh P. R. P. Verma Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India
antibacterial properties. This will not only avoid unnecessary introduction of inactive excipients in the body, but will also reduce the dose of antibiotics because of synergistic effects of excipients and drug acting together. Keywords Atomic force microscopy Scanning electron microscopy Transmission electron microscopy Pseudomonas aeruginosa Nanoemulsions
Introduction Pseudomonas aeruginosa is a widespread environmental Gram-negative bacillus that acts as an opportunistic pathogen under several circumstances (Lyczak et al. 2000). It has been implicated in a broad range of infections, such as endocarditis, folliculitis, keratitis, meningitis, pneumonia, urinary tract infections and wound infections (Henriques et al. 2011). In health care settings, the bacterium is an important cause of infection in vulnerable individuals including those with burns or neutropenia or receiving intensive care. In these groups’ morbidity and mortality attributable to P. aeruginosa can be high (Kerr and Snelling 2009). Management of infections associated with this bacterium is difficult due to the continued emergence of antibiotic resistance (Kerr and Snelling 2009). P. aeruginosa are known for their very low non-specific permeability and/or the presence of
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membrane-associated energy-driven efflux systems that makes them resistant to the majority of antibiotics (Sachetelli et al. 2000). Other mechanisms of resistance, like enzymatic inactivation of the drug and the alteration of the molecular target, can also work synergistically with these mechanisms (Sachetelli et al. 2000). Very recently, an outbreak of carbapenem-resistant P. aeruginosa infection in a surgical intensive care unit has been reported (Kohlenberg et al. 2010). Moreover, with the exception of doripenem, no new anti-Pseudomonas drugs have reached the market in recent years (Page and Heim 2009). Under this backdrop, an undeniable and imperative requirement to develop new effectual therapeutic approaches to contain the bacterium is self-evident. To address this, we thought to employ particular amalgamation of specific lipids that hold an innate antibacterial activity and which can be transformed into nanoemulsions for hosting and ferrying the antibiotics. The fundamental inspiration was to confer a concerted antibacterial activity of lipidic nanoemulsions and the drug (if ladened) working in tandem. Pharmaceuticalli, it is well established that to deliver any drug a proper combination of pharmaceutically inactive excipients are required. However, to deliver antibiotics the delivery approaches needs to be reassessed by using certain excipients that possess inherent antibacterial properties. This will not only avoid unnecessary introduction of inactive excipients in the body with but will also reduce the dose of antibiotics because of synergistic and tandem effect of excipients and drug acting together. Nanoemulsions doped with antibiotics have been explored by numerous researchers for their bactericidal activity. In a study, injectable benzathine-Penicillin G nanoemulsions were prepared and studied for their physicochemical and antimicrobial activities (Santos-Magalhaes et al. 2000). Nanoemulsions have been shown to be a stable intravenous formulation of rifampicin (Ahmed et al. 2008). Nanoemulsion made of soybean oil, Triton X-100 detergent, and tri-n-butyl phosphate in 20 % water has been shown to exhibit efficient sporicidal activity against a variety of Bacillus spores, including Bacillus anthracis (Hamouda et al. 1999). In a study, nanoemulsions comprising of Triton X-100 (10 % v/v), soybean oil 25 % v/v, and cetylpyridinium chloride (1 % w/v) showed an effectual activity against the antibiotic-resistant forms of Acinetobacter baumannii with overall activity
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remaining unchanged when exposed to severe environmental condition simulating field conditions of Iraq and Afghanistan (Hwang et al. 2013). Dapsone loaded nanoemulsion has been shown to be a promising system for the topical delivery and for the treatment of acne and leprosy (Borges et al. 2013). A water-in-oil emulsion technique has been established for preparing nanoemulsion of chitosan/heparin with better encapsulation of amoxicillin (Lin et al. 2012) An oil-in-water submicron emulsion of ciprofloxacin with sodium deoxycholate showed enhanced antimicrobial efficacy against Escherichia coli, Staphylococcus aureus, and P. aeruginosa in vitro (Jain et al. 2011). Nanoemulsions have been shown to inactivate suspensions of vegetative cells of Salmonella spp., E. coli, P. aeruginosa, S. aureus and Listeria monocytogenes (Teixeira et al. 2007). Furthermore, nanoemulsions formulated with different oils (devoid of any antibiotic drug) have also been found to be effective antibacterials, for example, peppermint oil nanoemulsion (Liang et al. 2012) cinnamon oil nanoemulsion (Ghosh et al. 2013) and eucalyptus oil nanoemulsion (Sugumar et al. 2014). Overall, results of these studies suggest that antibacterial activity of oils could be enhanced by dispensing them into nano form. A detailed application of nano-engineered drug delivery system for antibiotic therapy has been recently reviewed by Kalhapure et al. (2015). Lipids such as fatty alcohols, free fatty acids and monoglycerides of fatty acids are known to be potent antimicrobial/microbicidal agents in vitro and to kill enveloped viruses, Gram-positive and Gram-negative bacteria and fungi on contact (Thormar and Hilmarsson 2007). However, no pharmaceutical products containing lipids as active compounds have as yet been approved for clinical use as prophylactic or therapeutic drugs against bacterial and viral infections, even after the proven antimicrobial activities of lipids (Thormar and Hilmarsson 2007). Apparently, the great success of chemotherapy using synthetic antibiotics against bacterial and fungal infections and nucleoside analogues against viral infection has discouraged researchers and the pharmaceutical industry in making serious efforts to develop drug containing simple natural compounds (Thormar and Hilmarsson 2007). In our preceding studies, blank lipidic nanoemulsions (cationized and non-cationized) were prepared and their antibacterial activities were evaluated against an opportunistic bacterium Escherichia coli (Singh
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et al. 2015a, b) and Staphylococcus aureus (Singh et al. 2014). The placebo lipidic nanoemulsions were characterized by their globule size using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Ultimately, antibacterial mechanism of nanoemulsions was elucidated by TEM, atomic force microscopy (AFM) and scanning electron microscopy (SEM) (Singh et al. 2015a, b, 2014). In this study, the anti-bacterial activities of lipidic nanoemulsions were elucidated by TEM, SEM, and AFM against P. aeruginosa. The instrumental results were further corroborated by the quantitative studies such as zone of inhibition studies, cytoplasmic release studies, percent viable colonies and minimum inhibitory concentration studies.
Materials and methods Capmul MCM C8 (CMCM8) and Labrasol (LBS) was kind contribution from Abitec Corporation (Janesville, Germany) and Gattefosse (Saint-Priest, Cedex, France), respectively. Cremophor RH40 (CRH40) was a generous gift from BASF (Ludwigshafen, Germany). Oleyl amine (OA) was procured from Fluka (Steinheim, the Netherlands). P. aeruginosa (MTCC-647) was purchased from Microbial Type Culture and Gene Bank (IMT, Chandigarh, India). Preparation of lipidic emulsions Non-cationized nanoemulsion (NCN) was formulated by analogous method described by us before (Singh et al. 2015a, b). Briefly, NCN was prepared by taking CMCM8, LBS and CRH40 in the ratio of (1:1:2) in a sterilized beaker and warmed on a water bath with constant stirring till homogenous mixture was formed. The pre-heated Millipore water (100 mL) kept at the same temperature was added very slowly with constant stirring to get a clear NCN. The cationized nanoemulsion (CNE) (100 mL) was formulated by a similar procedure described above with the inclusion of 0.5 % w/w of OA. Morphological studies Transmission electron microscopy studies TEM study was done to study the structural organization of control cells and to explore the
anti-P. aeruginosa activities of NCN and CNE. The bacterial culture was exposed to two different treatments as mentioned before with minor adjustments (Singh et al. 2014). Briefly, the bacterium was cultured in 100.0 mL nutrient broth (NB) at 37 °C in an incubator for overnight to obtain exponential growth phase (*105 cfu/mL). The cultures were then centrifuged at 30009g for 30 min followed by washing thrice with sterile water for injection (SWI) to remove adhered salts or media that may meddle with TEM images. The harvested cells were then diluted with 5.0 mL of SWI followed by treatment with NCN (1.0 mL) and CNE (1.0 mL) separately, for 60 min. The samples treated with CNE and NCN were then equivalently (3.0 mL) separated in four different sterilized micro centrifuge tubes (coded as CNE-1, NCN-1, CNE-2, and NCN-2). From the first set (CNE-1 and NCN-1), a drop of treated bacterial specimen, were spread on a copper grid coated with carbon film separately, and a drop of 2.0 % w/v phosphotungstic acid solution was dripped on the copper grid. After 60 s, excess of solution was then removed by filter paper and grid was then air-dried at room temperature before loading in the TEM. This treatment ensures the presence of nanoemulsions in sample to explore the effect on CNE and NCN on the bacterial surface. The second set (CNE-2 and NCN-2) was centrifuged again at 30009g for 30 min to get bacterial pellets. The supernatant containing CNE and NCN was discarded followed by washing (two times) to warrant removal of unattached nanoemulsions. A drop of nanoemulsion free treated bacterial specimen (CNE and NCN) was visualized using TEM by the similar method as described above. Atomic force microscopy studies P. aeruginosa were treated with NCN and CNE by the similar method as reported under TEM studies. Care was taken to remove salts, nutrient media and lipidic nanoemulsions (CNE and NCN) after treatment to ensure no interference during AFM analysis. The bacterial samples (control, NCN and CNE treated; 10.0 lL) were placed over cover slips previously treated with poly-L-Lysine and kept at room temperature overnight. Atomic force microscopy (AFM) studies were performed at room temperature, using NT MDT Solver Pro 47 (Russia) in non-contact mode,
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using commercial silicon nitride probes (NT MDT, Russia) with following specifications: (a) cantilevers (100 mm); (b) spring constants (420 N/m); (c) tip curvature radius (5–10 nm) and (d) scan rate of 0.85 Hz. Dimensional analyses (DA) and contour profile (CP) were performed using the Solver Pro Nova program (NT MDT, Russia) of the system. Root mean square (rms) roughness of central part of cell (control as well as NCN and CNE treated) was calculated using the same programme after first order flattening to reduce the influence of cell curvatures at the edges of the image (Deupree and Schoenfisch 2009). Scanning electron microscopy P. aeruginosa bacterial samples (control, CNE and NCN treated) were prepared on sterilized cover slips by the similar method as reported for AFM analysis. The SEM was carried out with JEOL, JSM-6390LV, Japan. Prior to the examination, the samples were platinum sputter-coated to render them electrically active (Singh et al. 2015b). Agar diffusion and cytoplasmic release studies Agar diffusion studies were conducted to conclude the antibacterial activity of NCN and CNE against P. aeruginosa. 1000 lL of bacterial suspension was poured and spread aseptically on solidified nutrient agar. After inoculums absorption, wells were made using sterile tubes and were filled with 100.0 lL of the NCN and CNE followed by incubation at 37 °C for 24 h. The inhibition zones were computed using vernier callipers. This experiment was carried out in triplicate. The killed P. aeruginosa will bleed cytoplasmic contents (DNA and RNA) in the media which can be examined by taking absorbance at 260 nm (Xing et al. 2009). Bacterial cultures grown overnight in NB were harvested by centrifugation at 30009g for 30 min followed by washing and re-suspending in 0.5 % w/v sodium chloride solution. The suspension was attuned to an absorbance of 0.7 at 420 nm (Lin et al. 2012). A two mL portion of CNE and NCN was mixed with an equivalent volume of bacterial suspension and incubated for 90 min. The cell constituent released in the supernatant was examined by measuring UV absorption at 260 nm using suitable dilution.
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Determination of percent viable colonies The killing kinetics of NCNs and CNEs was performed by challenging 5.0 mL (*105 cfu/mL) of bacterial culture with 1 mL of CNEs and NCNs separately in a sterile test tube followed by incubation for 60 min at 37 °C. Control samples were prepared by taking 1.0 mL Millipore water instead of CNE or NCN. For viable counts, 0.5 mL of treated sample was taken from each labelled tube at varied time intervals and was mixed with sterilized molten NA plates. After solidification, the NA plates were incubated at 37 °C for 24 h in an inverted position. The experiment was performed in triplicate for each set of conditions. The percent inhibition of microbial count at different period was calculated by number of colonies found on treated plates divided by that of the control blank. The experiment was carried out by the method as reported previously (Singh et al. 2014; Zhang et al. 2010). Determination of minimum inhibitory concentration using resazurin microtiter-plate method The minimum inhibitory concentration (MIC) of NCNs and CNEs was evaluated using modified Resazurin microtitre-plate (96 well plate) method, as reported previously with some modifications (Sarker et al. 2007). The assay was carried out by adding 50 lL of NB in all wells of first and second rows. Then after, 50 lL of NCN and CNE was added in the first well of first and second row respectively. Serial dilutions were carried out until all the wells contained 50 lL of the solutions. 5 lL of resazurin indicator solution were added in each wells, followed by the addition of 5.0 lL (*105 cfu/mL) of bacterial suspension. The concentration in each well in descending concentration was 90.91, 45.46, 22.73, 11.37, 5.70, 2.85, 1.43, 0.72, 0.36, 0.18, 0.09, 0.045 mg/mL. The last two rows served as a positive (to confirm the viability of bacterial culture, without samples) and negative control (to confirm sterility of working conditions and solutions). The Microplates were securely wrapped with aluminium foil to prevent any contamination. Plates were prepared in triplicate, and incubated at 37 °C for 24 h. The color changes were observed by visual inspection. Blue color indicated absence of microorganism, while pink color indicated the growth of P. aeruginosa. The lowest concentration
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showing color change from blue to pink was considered as MIC value.
Results and discussion Morphological studies The representative TEM images from different areas of the same copper grid of untreated (control) P. aeruginosa are depicted in Fig. 1a, b and 2a, b. The cells appeared normal rod-shaped with smooth continuous double membrane with no visible cell damages (like pores, holes, grooves or breakages in cell envelope) indicating good preservation of cells during sample preparation. The present finding is in concurrence with the study carried out by Zhou et al. (2011). The continuous double membrane comprising of cell wall (CW) and plasma membrane (PM) of bacterial cell is demonstrated in Fig. 1b (shown by arrows). In one of the image (Fig. 2b), a cell dividing from middle into two halves owing to the creation of septum was observed (shown by arrow). The representative TEM images of NCN treated P. aeruginosa from different grid regions to demonstrate the full range of associated morphologies under the first proviso (i.e., when nanoemulsions were not removed after treatment) is illustrated Fig. 1c–h. Spherical non-cationized nanoemulsions were abundantly seen all around the bacterial cells (black square of Fig. 1c–h) and also copious numbers of nanoemulsions can be seen adhered to the bacterial cell wall (shown by black arrows in Fig. 1c–e, h). The compilation of TEM morphologies (Fig. 1c–h) of NCN treated P. aeruginosa showed various levels of cell disintegration exemplified by (1) the formation of blebs (cellular parts of cell attached to cell surface) shown by red arrows (Fig. 1c–d, g); (2) discontinuity and breakage of cell wall at various points (shown by blue arrows, Fig. 1c–f, h) followed by exudation of essential cellular contents; (3) debris of amorphous cellular contents present near to the vicinity of cells (shown within red square box, Fig. 1d, g, h); (4) loss of characteristics contour of cell: from even and uniform to peculiarly wavy and undulating with protuberances and finally leading to cell death. In an effort to identify the presence of NCN on the surface of bacterial cells after removing the NCN post treatment, bacterial cells were treated by the second approach as discussed
under materials and method section. It was observed that non-cationized nanoemulsions were not present on the surface of bacterial cells (supplemental Fig. S1a, c, e). This might be due to weak adherence forces between negatively charged bacterial surface (because of lipo-polysaccharides) and negatively charged NCN. The NCN possess a negative charge of -10.8 ± 0.80 mV as reported in our previous publication (Singh et al. 2015a). The lethal effect of NCN is evident from the presence of irregular and broken cell wall accompanied with blebs (shown by blue arrows), and exudation of essential cellular materials (shown within red dotted square) (supplemental Fig. S1a, c, e). The simultaneous existence of dead cells and absence of NCN on cell surface signifies the internalization of NCNs through the bacterial membranes (lipo-polysaccharides cell wall and plasma membrane) into the cytoplasm of cells causing irretrievable damage to cells and ultimately causing cell death. The TEM illustrations of CNEs treated P. aeruginosa from different grid regions (when CNEs were not removed after treatment) are exemplified in Fig. 2c–h. As examined with the NCNs treated cells, the spherical cationized nanoemulsions are distributed uniformly in copious numbers at the vicinity and on the bacterial surface. The representative unadhered CNEs are shown within black squares while adhered CNEs are shown by black arrows (Fig. 2c– h). In accordance to the NCNs treated cells, the assemblage of TEM morphologies (Fig. 2c–h) of CNEs exposed P. aeruginosa demonstrate different levels of cell injury symbolized by following events: (1) bleb formation shown by red arrows (Fig. 2c, e, g, h); (2) breakage of cell wall at various points (indicated by red double end arrow; Fig. 2e–f) pursuing exudation of essential cytoplasmic contents leading to accumulation of cytoplasmic debris in proximity of cells (shown within black dotted box, Fig. 2d, f); and (3) abnormal and wavy cell contour with occasional protuberances. Furthermore, the analysis of Fig. 2d (enlarged portion of Fig. 2c) reveals some interesting facts: the area within red dotted box (Fig. 2d) appears to be cytoplasm oozing out from an opening created in lipopolysaccharide (LPS) layer (the opening is supposed to be of sufficient depth extending deep into plasma membrane) in thin stream and making a pool of cytoplasm at the vicinity of cells (shown within black dotted box, Fig. 2d). The path of oozing
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Antonie van Leeuwenhoek Fig. 1 Transmission electron microscopic images of untreated (a–b) and noncationized nanoemulsions (NCN) treated P. aeruginosa cells (c–h). The arrows (b) illustrate the presence of smooth cell wall (CW) and plasma membrane (PM) of untreated bacteria. Black squares indicate the presence of non-adhered NCN (c–h). Black arrows (c–e, h) indicate the NCN attached to bacterial cell wall. Red arrows (c–d, g) and blue arrows (c–f, h) show blebs formation and breakage of cell wall at various points, respectively. The exudations of essential cellular contents at vicinity of cells are shown within red square box (d, g–h). The photomicrographs were taken from different grid areas. Scale bar (a– f) 1000 nm; (g) 2000 nm; (h) 500 nm
cytoplasm leading to the pool of cytoplasm is demonstrated by the red curve arrow (Fig. 2d). The cell wall of this area is witnessed by occurrence of numerous CNEs attached on the surface of bacteria (shown by black arrow). Another interesting finding is that the disintegration of cell wall is not taking place in one particular place or point but rather it is occurring simultaneously throughout out the bacterial surface as shown by red dotted double end arrows (Fig. 2e, f). It appears that CNEs have high
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cell-membrane piercing potential and their foremost biocidal mechanism might be fusion and followed by membrane commotion. The kind of killing mechanism relies more on collision frequency between nanometric CNEs and bacterial cells followed by diffusion through the cell membrane driven by a concentration gradient since CNEs is present in high concentration outside the bacterial surface. Similar findings were also reported by Chang et al., (2012). Furthermore, it is also apparent that the thickness of
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Fig. 2 Transmission electron microscopic images of untreated (a–b) and cationized nanoemulsions (CNE) treated P. aeruginosa cells (c–h). In one of the image (b), a cell dividing from middle into two halves owing to the creation of septum was observed (shown by arrow). The un-adhered CNE and adhered CNE are shown within black squares (c–h) and black arrows (c– h) respectively. The bleb formations are shown by red arrows
(c, e, g–h) and breakage of cell wall at various points are shown by red double end arrow (e–f). The exudations of essential cytoplasmic contents are shown within black dotted box (d, f). The path of oozing cytoplasm leading to formation of cytoplasm’s pool is demonstrated by red curve arrow (d) and red dotted box (d). The photomicrographs were taken from different grid areas. Scale bar a–b, g 1000 nm; c, h 500 nm; d–f 200 nm
cell wall gradually decreases due to the constant bombardment of numerous CNEs. The reduced thickness of cell wall further enhances the diffusion
of CNEs as per Fick’s law of diffusion that state that the rate of diffusion is indirectly proportional to the thickness of the membrane.
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Cationized lipidic nanoemulsions possess a positive zeta potential of ?20.9 ± 0.46 mV as reported by us previously (Singh et al. 2015a). TEM images of CNEs treated bacterial cell followed by removal of CNEs post treatment indicated no adherence of CNEs on the bacterial surface (supplemental Fig. S1b, d, f). The finding is in contrary to our previous studies with S. aureus where the CNEs remained adhered to the Gram-positive bacteria (Singh et al. 2014). The present findings remains un-answered and requires further studies by incorporating various amounts of oleylamine and making series of CNEs with different degrees of zeta potential which may give optimal value for more efficient electrostatic attractions between negatively charged bacteria. Furthermore, the process of removing CNEs could be carried out at lesser agitation stress so as to observe the presence of electrostatic attractions between oppositely charged cell wall and CNEs. Nevertheless, mild electrostatic attractions between the bacterial cells and CNEs cannot be ruled out. The bactericidal effect of CNEs is evident from supplemental Fig. S1b, d, f that showed the presence of blebs (blue arrows), exudation of cytoplasmic contents from neighbouring cells giving slimy appearance (shown within red square), and deterioration of LPS layer. It is a well known fact that the LPS layer of Gram-negative bacteria acts as a permeability barrier providing stability to the outer membrane and restrict the entry of any foreign particles that might kill or injure the bacteria. Moreover, LPS also contribute to the bacterial attachment to the surface and biofilm formation, apart from providing bacterial resistance to antimicrobial agents (Sotirova et al. 2009). Therefore, it very much evident that turbulence caused by CNEs to the LPS layer will certainly lead to bacterial lysis causing reduced bacterial resistance. The anti-P. aeruginosa effects of NCNs and CNEs were additionally substantiated by SEM studies. The SEM images of control bacteria showed regular rodshaped morphology with smooth cell wall without any structural defects (Fig. 3a, b). However, the NCNs and CNEs treated cells showed evidence of bactericidal effects as shown in Fig. 3c, d and 3e, f, respectively. The extent of cell damage was more with CNEs treated cells as compared to NCNs treated cells. CNEs treated cells had lost the structural integrity with disintegration of cell wall, and leaving behind fragmented residues of cells (shown within red dotted square of
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Fig. 3f). This might be due to the mild electrostatic interaction between cationized nanoemulsions and anionic P. aeruginosa. The negative charge on bacterial surface is mainly due to anionic biophosphorylated sugar head groups of LPS as well as negatively charged lipids like cardiolipin (Ouberai et al. 2011). The enhanced electrostatic interactions lead to the accumulation of CNEs within membrane followed by destabilization of lipid membrane. To further corroborate the comparative efficiency of antibacterial activities of these nanoemulsions (NCNs and CNEs), atomic force microscopy was performed. The AFM images of control P. aeruginosa cells are depicted in Figs. 4a–d and 5a, b. Figure 4a, b portrays 2-D and 3-D AFM images of untreated cells while Fig. 4c, d depicts the contour profile (CP) and dimensional analysis (DA) of the same bacteria. Similarly, Fig. 5a, b illustrates the 2D and 3D AFM images of untreated bacterial cells from different sectional area. These AFM images illustrate the smooth, homogenous and unbroken surface topology in all directions of bacteria with no signs of cell fragmentation. The root mean square (rms) roughness of untreated bacteria was 1.74 ± 0.07, indicating smooth surface sufficing the DA and CP images. The 2-dimensional atomic force microscopic images of NCNs treated cells are portrayed in Fig. 4e while its 3D construction and contour profile is depicted in Fig. 4f and 4g, respectively. The rms roughness P. aeruginosa cells exposed to NCNs treated were found to increase from 1.74 ± 0.07 (control) to 12.7 ± 2.51 nm, demonstrating increased cell roughness. The enhanced rms roughness can further be corroborated from contour profile (Fig. 4g) that showed numerous fragmented remains of bacteria (shown by arrows). The increased roughness might be because of binding of NCNs with lipopolysaccharide (LPS) layer causing the progressive erosion and thereby exposing more roughed thin peptidoglycan (PDG) layer lying beneath to it. The diminished thickness of LPS layer may further facilitate the passage of copious NCNs invading the PDG layer. The NCN may further destabilize the PDG and cellmembrane and may lead to the release of cytoplasmic content containing genetic materials causing fragmentation of bacteria and cell death. The analysis of Fig. 4e–g further reveals that the overall thickness of the cell was diminished sufficing irreparable loss to bacteria because of exudation of cytoplasmic content.
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Fig. 3 Scanning electron microscopy images of untreated P. aeruginosa cells (a–b), non cationized treated (c–d) and cationized treated (e–f). Area within red dotted square indicates the dead residues of bacterial cell when treated with cationized lipidic emulsions
The finding is in accordance with the TEM studies as discussed earlier. Figure 5c–e portrays the 2D, 3D and CP AFM images of CNEs treated P. aeruginosa. The rms roughness of the CNEs treated P. aeruginosa cells was 22.16 ± 3.14 nm, indicative of extensive cell roughness as compared to control (1.74 ± 0.07 nm) and NCN treated cells (12.7 ± 2.51 nm). The enhanced rms was substantiated by the analysis of contour profile (Fig. 5e), where extensive damage was apparent with cell flattening, and relatively more occurrence of fragments as compared to NCNs treated cells (compare Figs. 4g and 5e). The comparatively enhanced fragmentation with increased rms of CNEs treated cell can be ascribed to the mild electrostatic
attraction between positively charged CNE and negatively charged bacterial surface that renders better anchoring to the LPS layer causing successive damages to PDG and cell membrane causing the exudation of essential cytoplasmic contents. Furthermore, it has been reported that the disruption of LPS layer witnessed by increased surface roughness provides indirect evidence that the protein and/or DNA damage contributes significantly to the cytotoxic effects against P. aeruginosa (Deupree and Schoenfisch 2009). In addition to the nanosized droplets and cationization of lipidic emulsions, the composition of lipidic emulsions that includes blends of fatty acids (caprylic acid, caproic acid, lauric acid etc.,) may further enhance the anti-Pseudomonas activities. Any
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Fig. 4 Atomic force microscopic images of untreated (a–d) and non-cationized nanoemulsions (NCN) treated P. aeruginosa cells (e–g). 2-D (a) and 3-D (b) AFM images of untreated bacteria showed no cell deformation while contour profile (c) and
dimensional analysis (d) demonstrates the smooth surface of bacteria without any cell damages. 2-D (e); 3-D (f) and contour profile (g) images of NCN treated bacterial cells showed bacterial fragmentation (shown by arrows, g) leading to cell lysis
molecule that has to traverse through the LPS and plasma membrane layer of Gram-negative bacteria should have optimum hydrophilic-lipophilic balance,
i.e., it should be amphiphilic in nature (having polar and non-polar groups). This is because the LPS and plasma-membrane layer contains polar moieties
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Fig. 5 Atomic force microscopic images of untreated (a– b) and cationized nanoemulsions (CNE) treated P. aeruginosa cells (c–e). 2-D (a) and 3-D (b) AFM images of untreated bacteria showed no cell deformation. 2-D (c); 3-D (d) and
contour profile (e) of CNE treated bacterial cells showed extensive bacterial fragmentation (shown by arrows, e) as compared to NCN treated bacterial cells
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(ethanolamine) that provides hydrophilicity, and nonpolar moieties (fatty acid chains) that imparts hydrophobicity to the rate-limiting barrier. If the molecule is hydrophobic in nature then it may retain only in non-polar part of LPS and will be unable to move forward. Similarly, if the molecule is extremely hydrophilic than it may get concentrated only in the hydrophilic domains and cannot move further deeper into the layer. In the present study, we have selected CMCM8 which is chemically glyceryl mono caprylate (GMC) having HLB value of 4.7, and labrasol which is chemically caprylocaproyl polyoxyl-8 glycerides (CPG) with HLB value of 12, to prepare NCNs and CNEs. Mixed HLB system incorporated into nanoemulsions can efficiently traverse through the LPS and plasma-membrane causing irreparable cell death. Additionally, CPG have been reported to be potent P-pg inhibitor (Lin et al. 2007). It is noteworthy that a major mechanism of P. aeruginosa resistance is the presence of efflux pump embedded in LPS layer that does not allow the entry of drug (Sotirova et al. 2009). CPG being efflux inhibitor may lead to reduced drugresistant of the bacteria.
damage, which is in agreement with the TEM, SEM, and AFM studies.
Agar diffusion and cytoplasmic release studies
Resazurin is an oxidation–reduction indicator used for the evaluation of cell-growth, particularly in various cytotoxic assays (Sarker et al. 2007). It is a blue (weakly fluorescent) and non-toxic dye that turns to pink, when reduced to resorufin by oxidoreductases within viable cells. The MIC was thus determined visually at lowest concentration when no color changes occurred. The representative 96 well microtiter plate showing MIC of NCN and CNE is illustrated in supplemental Fig. S3. The MIC of NCN and CNE was found at 11.37 and 5.70 mg/mL, respectively, demonstrating enhanced anti-Pseudomonas activity of CNE as compared to NCN. The present finding is in accordance with the literature where the positively charged niosomes resulted in the significant reduction in MIC compared to the noncharged niosomes (Abdelaziz et al. 2015). In an another study effectiveness of cationic nanoemulsions (chitosan-coated nanoemulsion) was demonstrated as therapeutics to intervene E. coli induced peritonitis as well as in sepsis (Jain et al. 2014). Furthermore, in a study antimicrobial nanoemulsions were prepared using thyme oil and were cationized by lauric arginate that showed enhanced anti-microbial activity as compared to non-cationized emulsion (Chang et al. 2015).
The representative zone of inhibition exhibited by CNEs (black dotted circle) and NCNs (red dotted circle) against P. aeruginosa is illustrated in supplemental Fig. S2. The inhibition zone was found to be 14.3 ± 1.15 and 10.3 ± 0.6 mm for CNEs and NCNs, respectively. The enhanced inhibition zone exhibited by CNEs can be attributed to mild to moderate adhesion of cationized nanoemulsion with negatively charged bacteria sufficing the findings of TEM, SEM and AFM. The release of UV-absorbing cytoplasmic content from the P. aeruginosa exposed to CNEs and NCNs is an indicator of changes in membrane permeability of bacteria. Bar graph shown in Fig. 6 indicated significant (p \ 0.05) increase of cytoplasmic material when bacterial cells were treated with NCN (0.234 ± 0.023) and CNE (0.425 ± 0.077) as compared to control (0.024 ± 0.010). The t test applied to the mean absorbance values of CNE and NCN treated cells, revealed significant increase in cytoplasmic content in CNE treated cells as compared to NCN (p \ 0.05) illustrating that the CNEs had caused considerable membrane permeation leading to release of cytoplasmic content causing permanent cell
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Percent viable colonies Percent viable colonies of P. aeruginosa remaining after treatment with NCNs and CNEs were determined in time-dependent manner in specific conditions of nutrient media, temperature and time and the results are portrayed in Fig. 6. The control and untreated bacterial cells showed no decline in percent viable colonies at different time intervals (Fig. 6). When bacterial cells were exposed to NCN and CNE, the viable colonies decreased in time-dependent manner. The rate of decline was found higher in CNE as compared to NCN with no observed viable colonies at 60 min (NCN treated) and 15 min (CNE treated). The finding can be attributed to the enhanced adhesion of CNE with bacterial cell walls as compared to NCN, which is in agreement with the previous findings of zone of inhibition and cell constituent release studies. Determination of minimum inhibitory condition
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Fig. 6 Percent viable colonies of P. aeruginosa remaining when treated with non-cationed nanoemulsion (NCN) and cationized nanoemulsion (CNE) at varied time intervals. Bar graph indicates the release of cytoplasmic content exhibited by absorbencies at 260 nm when P. aeruginosa were treated for 1 h with NCN and CNE
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The findings further support the results of morphological studies (TEM, SEM, and AFM) as well as other in vitro anti-Pseudomonas studies (agar diffusion studies, cell constituent studies and time-dependent killing studies)
Conclusions The present study demonstrates the anti-Pseudomonas activities of cationized and non-cationized nanoemulsions using transmission/scanning electron microscopy and atomic force microscopy. The microscopic studies were corroborated by in vitro studies such as zone of inhibition, cytoplasmic release studies, time-dependent killing studies and MIC studies. The microscopic studies demonstrated the cell-lysis due to the formation of blebs, exudation of essential cellular contents and loss of characteristic contours of the cells. The in vitro anti-Pseudomonas studies further supported the findings of microscopic studies. The enhanced inhibition exhibited by cationized nanoemulsions can be attributed to mild to moderate adhesion of cationized nanoemulsion with negatively charged bacteria. In conclusion, it can be inferred that the delivery issues of antibiotics could be reassessed by using lipids that possess inherent antibacterial properties. This will
reduce the dose of antibiotics because of synergistic and tandem effect of excipients and drug acting together. Acknowledgments Authors acknowledges University Grant Commission (UGC), Government of India for providing the financial assistance vide sanction letter number ‘‘41-1423/2012 (SR), dated 30 July 2012, to Mrs. Neeru Singh. The constructive comments and suggestion of anonymous reviewers is gratefully acknowledged. Conflicts of interest Authors report no conflict of interest. The authors alone are responsible for the contents and writing of the paper. Research involving Human participants and/or animals This article does not contain any studies with human participants or animals performed by any of the authors.
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ORIGINAL PAPER
Evidence for bactericidal activities of lipidic nanoemulsions against Pseudomonas aeruginosa Neeru Singh . Saurabh Manaswita Verma . Sandeep Kumar Singh . Priya Ranjan Prasad Verma
Received: 18 November 2014 / Accepted: 4 April 2015 Ó Springer International Publishing Switzerland 2015
Abstract Pseudomonas aeruginosa has been implicated in a broad range of infections and shown to acquire rapid resistance to anti-microbial agents. In the present, study we have used particular amalgamation of specific lipids that hold innate antibacterial activities, which can be transformed into cationized and non-cationized nanoemulsions. The anti-Pseudomonas activities were then elucidated by transmission/scanning electron microscopy, and atomic force microscopy. The microscopic studies revealed the cell lysis due to the formation of blebs, exudation of essential cellular contents and loss of characteristics contour of the cells. The microscopic studies were then corroborated by zone of inhibition, cytoplasmic release studies, time dependent killing and MIC determination. Conclusively, it can be inferred that the delivery issues of antibiotics could be reassessed by using certain excipients that possess inherent
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0449-8) contains supplementary material, which is available to authorized users. N. Singh (&) Division of Biomedical Lab Technology, University Polytechnic, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India e-mail: [email protected] S. M. Verma S. K. Singh P. R. P. Verma Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India
antibacterial properties. This will not only avoid unnecessary introduction of inactive excipients in the body, but will also reduce the dose of antibiotics because of synergistic effects of excipients and drug acting together. Keywords Atomic force microscopy Scanning electron microscopy Transmission electron microscopy Pseudomonas aeruginosa Nanoemulsions
Introduction Pseudomonas aeruginosa is a widespread environmental Gram-negative bacillus that acts as an opportunistic pathogen under several circumstances (Lyczak et al. 2000). It has been implicated in a broad range of infections, such as endocarditis, folliculitis, keratitis, meningitis, pneumonia, urinary tract infections and wound infections (Henriques et al. 2011). In health care settings, the bacterium is an important cause of infection in vulnerable individuals including those with burns or neutropenia or receiving intensive care. In these groups’ morbidity and mortality attributable to P. aeruginosa can be high (Kerr and Snelling 2009). Management of infections associated with this bacterium is difficult due to the continued emergence of antibiotic resistance (Kerr and Snelling 2009). P. aeruginosa are known for their very low non-specific permeability and/or the presence of
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membrane-associated energy-driven efflux systems that makes them resistant to the majority of antibiotics (Sachetelli et al. 2000). Other mechanisms of resistance, like enzymatic inactivation of the drug and the alteration of the molecular target, can also work synergistically with these mechanisms (Sachetelli et al. 2000). Very recently, an outbreak of carbapenem-resistant P. aeruginosa infection in a surgical intensive care unit has been reported (Kohlenberg et al. 2010). Moreover, with the exception of doripenem, no new anti-Pseudomonas drugs have reached the market in recent years (Page and Heim 2009). Under this backdrop, an undeniable and imperative requirement to develop new effectual therapeutic approaches to contain the bacterium is self-evident. To address this, we thought to employ particular amalgamation of specific lipids that hold an innate antibacterial activity and which can be transformed into nanoemulsions for hosting and ferrying the antibiotics. The fundamental inspiration was to confer a concerted antibacterial activity of lipidic nanoemulsions and the drug (if ladened) working in tandem. Pharmaceuticalli, it is well established that to deliver any drug a proper combination of pharmaceutically inactive excipients are required. However, to deliver antibiotics the delivery approaches needs to be reassessed by using certain excipients that possess inherent antibacterial properties. This will not only avoid unnecessary introduction of inactive excipients in the body with but will also reduce the dose of antibiotics because of synergistic and tandem effect of excipients and drug acting together. Nanoemulsions doped with antibiotics have been explored by numerous researchers for their bactericidal activity. In a study, injectable benzathine-Penicillin G nanoemulsions were prepared and studied for their physicochemical and antimicrobial activities (Santos-Magalhaes et al. 2000). Nanoemulsions have been shown to be a stable intravenous formulation of rifampicin (Ahmed et al. 2008). Nanoemulsion made of soybean oil, Triton X-100 detergent, and tri-n-butyl phosphate in 20 % water has been shown to exhibit efficient sporicidal activity against a variety of Bacillus spores, including Bacillus anthracis (Hamouda et al. 1999). In a study, nanoemulsions comprising of Triton X-100 (10 % v/v), soybean oil 25 % v/v, and cetylpyridinium chloride (1 % w/v) showed an effectual activity against the antibiotic-resistant forms of Acinetobacter baumannii with overall activity
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remaining unchanged when exposed to severe environmental condition simulating field conditions of Iraq and Afghanistan (Hwang et al. 2013). Dapsone loaded nanoemulsion has been shown to be a promising system for the topical delivery and for the treatment of acne and leprosy (Borges et al. 2013). A water-in-oil emulsion technique has been established for preparing nanoemulsion of chitosan/heparin with better encapsulation of amoxicillin (Lin et al. 2012) An oil-in-water submicron emulsion of ciprofloxacin with sodium deoxycholate showed enhanced antimicrobial efficacy against Escherichia coli, Staphylococcus aureus, and P. aeruginosa in vitro (Jain et al. 2011). Nanoemulsions have been shown to inactivate suspensions of vegetative cells of Salmonella spp., E. coli, P. aeruginosa, S. aureus and Listeria monocytogenes (Teixeira et al. 2007). Furthermore, nanoemulsions formulated with different oils (devoid of any antibiotic drug) have also been found to be effective antibacterials, for example, peppermint oil nanoemulsion (Liang et al. 2012) cinnamon oil nanoemulsion (Ghosh et al. 2013) and eucalyptus oil nanoemulsion (Sugumar et al. 2014). Overall, results of these studies suggest that antibacterial activity of oils could be enhanced by dispensing them into nano form. A detailed application of nano-engineered drug delivery system for antibiotic therapy has been recently reviewed by Kalhapure et al. (2015). Lipids such as fatty alcohols, free fatty acids and monoglycerides of fatty acids are known to be potent antimicrobial/microbicidal agents in vitro and to kill enveloped viruses, Gram-positive and Gram-negative bacteria and fungi on contact (Thormar and Hilmarsson 2007). However, no pharmaceutical products containing lipids as active compounds have as yet been approved for clinical use as prophylactic or therapeutic drugs against bacterial and viral infections, even after the proven antimicrobial activities of lipids (Thormar and Hilmarsson 2007). Apparently, the great success of chemotherapy using synthetic antibiotics against bacterial and fungal infections and nucleoside analogues against viral infection has discouraged researchers and the pharmaceutical industry in making serious efforts to develop drug containing simple natural compounds (Thormar and Hilmarsson 2007). In our preceding studies, blank lipidic nanoemulsions (cationized and non-cationized) were prepared and their antibacterial activities were evaluated against an opportunistic bacterium Escherichia coli (Singh
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et al. 2015a, b) and Staphylococcus aureus (Singh et al. 2014). The placebo lipidic nanoemulsions were characterized by their globule size using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Ultimately, antibacterial mechanism of nanoemulsions was elucidated by TEM, atomic force microscopy (AFM) and scanning electron microscopy (SEM) (Singh et al. 2015a, b, 2014). In this study, the anti-bacterial activities of lipidic nanoemulsions were elucidated by TEM, SEM, and AFM against P. aeruginosa. The instrumental results were further corroborated by the quantitative studies such as zone of inhibition studies, cytoplasmic release studies, percent viable colonies and minimum inhibitory concentration studies.
Materials and methods Capmul MCM C8 (CMCM8) and Labrasol (LBS) was kind contribution from Abitec Corporation (Janesville, Germany) and Gattefosse (Saint-Priest, Cedex, France), respectively. Cremophor RH40 (CRH40) was a generous gift from BASF (Ludwigshafen, Germany). Oleyl amine (OA) was procured from Fluka (Steinheim, the Netherlands). P. aeruginosa (MTCC-647) was purchased from Microbial Type Culture and Gene Bank (IMT, Chandigarh, India). Preparation of lipidic emulsions Non-cationized nanoemulsion (NCN) was formulated by analogous method described by us before (Singh et al. 2015a, b). Briefly, NCN was prepared by taking CMCM8, LBS and CRH40 in the ratio of (1:1:2) in a sterilized beaker and warmed on a water bath with constant stirring till homogenous mixture was formed. The pre-heated Millipore water (100 mL) kept at the same temperature was added very slowly with constant stirring to get a clear NCN. The cationized nanoemulsion (CNE) (100 mL) was formulated by a similar procedure described above with the inclusion of 0.5 % w/w of OA. Morphological studies Transmission electron microscopy studies TEM study was done to study the structural organization of control cells and to explore the
anti-P. aeruginosa activities of NCN and CNE. The bacterial culture was exposed to two different treatments as mentioned before with minor adjustments (Singh et al. 2014). Briefly, the bacterium was cultured in 100.0 mL nutrient broth (NB) at 37 °C in an incubator for overnight to obtain exponential growth phase (*105 cfu/mL). The cultures were then centrifuged at 30009g for 30 min followed by washing thrice with sterile water for injection (SWI) to remove adhered salts or media that may meddle with TEM images. The harvested cells were then diluted with 5.0 mL of SWI followed by treatment with NCN (1.0 mL) and CNE (1.0 mL) separately, for 60 min. The samples treated with CNE and NCN were then equivalently (3.0 mL) separated in four different sterilized micro centrifuge tubes (coded as CNE-1, NCN-1, CNE-2, and NCN-2). From the first set (CNE-1 and NCN-1), a drop of treated bacterial specimen, were spread on a copper grid coated with carbon film separately, and a drop of 2.0 % w/v phosphotungstic acid solution was dripped on the copper grid. After 60 s, excess of solution was then removed by filter paper and grid was then air-dried at room temperature before loading in the TEM. This treatment ensures the presence of nanoemulsions in sample to explore the effect on CNE and NCN on the bacterial surface. The second set (CNE-2 and NCN-2) was centrifuged again at 30009g for 30 min to get bacterial pellets. The supernatant containing CNE and NCN was discarded followed by washing (two times) to warrant removal of unattached nanoemulsions. A drop of nanoemulsion free treated bacterial specimen (CNE and NCN) was visualized using TEM by the similar method as described above. Atomic force microscopy studies P. aeruginosa were treated with NCN and CNE by the similar method as reported under TEM studies. Care was taken to remove salts, nutrient media and lipidic nanoemulsions (CNE and NCN) after treatment to ensure no interference during AFM analysis. The bacterial samples (control, NCN and CNE treated; 10.0 lL) were placed over cover slips previously treated with poly-L-Lysine and kept at room temperature overnight. Atomic force microscopy (AFM) studies were performed at room temperature, using NT MDT Solver Pro 47 (Russia) in non-contact mode,
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using commercial silicon nitride probes (NT MDT, Russia) with following specifications: (a) cantilevers (100 mm); (b) spring constants (420 N/m); (c) tip curvature radius (5–10 nm) and (d) scan rate of 0.85 Hz. Dimensional analyses (DA) and contour profile (CP) were performed using the Solver Pro Nova program (NT MDT, Russia) of the system. Root mean square (rms) roughness of central part of cell (control as well as NCN and CNE treated) was calculated using the same programme after first order flattening to reduce the influence of cell curvatures at the edges of the image (Deupree and Schoenfisch 2009). Scanning electron microscopy P. aeruginosa bacterial samples (control, CNE and NCN treated) were prepared on sterilized cover slips by the similar method as reported for AFM analysis. The SEM was carried out with JEOL, JSM-6390LV, Japan. Prior to the examination, the samples were platinum sputter-coated to render them electrically active (Singh et al. 2015b). Agar diffusion and cytoplasmic release studies Agar diffusion studies were conducted to conclude the antibacterial activity of NCN and CNE against P. aeruginosa. 1000 lL of bacterial suspension was poured and spread aseptically on solidified nutrient agar. After inoculums absorption, wells were made using sterile tubes and were filled with 100.0 lL of the NCN and CNE followed by incubation at 37 °C for 24 h. The inhibition zones were computed using vernier callipers. This experiment was carried out in triplicate. The killed P. aeruginosa will bleed cytoplasmic contents (DNA and RNA) in the media which can be examined by taking absorbance at 260 nm (Xing et al. 2009). Bacterial cultures grown overnight in NB were harvested by centrifugation at 30009g for 30 min followed by washing and re-suspending in 0.5 % w/v sodium chloride solution. The suspension was attuned to an absorbance of 0.7 at 420 nm (Lin et al. 2012). A two mL portion of CNE and NCN was mixed with an equivalent volume of bacterial suspension and incubated for 90 min. The cell constituent released in the supernatant was examined by measuring UV absorption at 260 nm using suitable dilution.
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Determination of percent viable colonies The killing kinetics of NCNs and CNEs was performed by challenging 5.0 mL (*105 cfu/mL) of bacterial culture with 1 mL of CNEs and NCNs separately in a sterile test tube followed by incubation for 60 min at 37 °C. Control samples were prepared by taking 1.0 mL Millipore water instead of CNE or NCN. For viable counts, 0.5 mL of treated sample was taken from each labelled tube at varied time intervals and was mixed with sterilized molten NA plates. After solidification, the NA plates were incubated at 37 °C for 24 h in an inverted position. The experiment was performed in triplicate for each set of conditions. The percent inhibition of microbial count at different period was calculated by number of colonies found on treated plates divided by that of the control blank. The experiment was carried out by the method as reported previously (Singh et al. 2014; Zhang et al. 2010). Determination of minimum inhibitory concentration using resazurin microtiter-plate method The minimum inhibitory concentration (MIC) of NCNs and CNEs was evaluated using modified Resazurin microtitre-plate (96 well plate) method, as reported previously with some modifications (Sarker et al. 2007). The assay was carried out by adding 50 lL of NB in all wells of first and second rows. Then after, 50 lL of NCN and CNE was added in the first well of first and second row respectively. Serial dilutions were carried out until all the wells contained 50 lL of the solutions. 5 lL of resazurin indicator solution were added in each wells, followed by the addition of 5.0 lL (*105 cfu/mL) of bacterial suspension. The concentration in each well in descending concentration was 90.91, 45.46, 22.73, 11.37, 5.70, 2.85, 1.43, 0.72, 0.36, 0.18, 0.09, 0.045 mg/mL. The last two rows served as a positive (to confirm the viability of bacterial culture, without samples) and negative control (to confirm sterility of working conditions and solutions). The Microplates were securely wrapped with aluminium foil to prevent any contamination. Plates were prepared in triplicate, and incubated at 37 °C for 24 h. The color changes were observed by visual inspection. Blue color indicated absence of microorganism, while pink color indicated the growth of P. aeruginosa. The lowest concentration
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showing color change from blue to pink was considered as MIC value.
Results and discussion Morphological studies The representative TEM images from different areas of the same copper grid of untreated (control) P. aeruginosa are depicted in Fig. 1a, b and 2a, b. The cells appeared normal rod-shaped with smooth continuous double membrane with no visible cell damages (like pores, holes, grooves or breakages in cell envelope) indicating good preservation of cells during sample preparation. The present finding is in concurrence with the study carried out by Zhou et al. (2011). The continuous double membrane comprising of cell wall (CW) and plasma membrane (PM) of bacterial cell is demonstrated in Fig. 1b (shown by arrows). In one of the image (Fig. 2b), a cell dividing from middle into two halves owing to the creation of septum was observed (shown by arrow). The representative TEM images of NCN treated P. aeruginosa from different grid regions to demonstrate the full range of associated morphologies under the first proviso (i.e., when nanoemulsions were not removed after treatment) is illustrated Fig. 1c–h. Spherical non-cationized nanoemulsions were abundantly seen all around the bacterial cells (black square of Fig. 1c–h) and also copious numbers of nanoemulsions can be seen adhered to the bacterial cell wall (shown by black arrows in Fig. 1c–e, h). The compilation of TEM morphologies (Fig. 1c–h) of NCN treated P. aeruginosa showed various levels of cell disintegration exemplified by (1) the formation of blebs (cellular parts of cell attached to cell surface) shown by red arrows (Fig. 1c–d, g); (2) discontinuity and breakage of cell wall at various points (shown by blue arrows, Fig. 1c–f, h) followed by exudation of essential cellular contents; (3) debris of amorphous cellular contents present near to the vicinity of cells (shown within red square box, Fig. 1d, g, h); (4) loss of characteristics contour of cell: from even and uniform to peculiarly wavy and undulating with protuberances and finally leading to cell death. In an effort to identify the presence of NCN on the surface of bacterial cells after removing the NCN post treatment, bacterial cells were treated by the second approach as discussed
under materials and method section. It was observed that non-cationized nanoemulsions were not present on the surface of bacterial cells (supplemental Fig. S1a, c, e). This might be due to weak adherence forces between negatively charged bacterial surface (because of lipo-polysaccharides) and negatively charged NCN. The NCN possess a negative charge of -10.8 ± 0.80 mV as reported in our previous publication (Singh et al. 2015a). The lethal effect of NCN is evident from the presence of irregular and broken cell wall accompanied with blebs (shown by blue arrows), and exudation of essential cellular materials (shown within red dotted square) (supplemental Fig. S1a, c, e). The simultaneous existence of dead cells and absence of NCN on cell surface signifies the internalization of NCNs through the bacterial membranes (lipo-polysaccharides cell wall and plasma membrane) into the cytoplasm of cells causing irretrievable damage to cells and ultimately causing cell death. The TEM illustrations of CNEs treated P. aeruginosa from different grid regions (when CNEs were not removed after treatment) are exemplified in Fig. 2c–h. As examined with the NCNs treated cells, the spherical cationized nanoemulsions are distributed uniformly in copious numbers at the vicinity and on the bacterial surface. The representative unadhered CNEs are shown within black squares while adhered CNEs are shown by black arrows (Fig. 2c– h). In accordance to the NCNs treated cells, the assemblage of TEM morphologies (Fig. 2c–h) of CNEs exposed P. aeruginosa demonstrate different levels of cell injury symbolized by following events: (1) bleb formation shown by red arrows (Fig. 2c, e, g, h); (2) breakage of cell wall at various points (indicated by red double end arrow; Fig. 2e–f) pursuing exudation of essential cytoplasmic contents leading to accumulation of cytoplasmic debris in proximity of cells (shown within black dotted box, Fig. 2d, f); and (3) abnormal and wavy cell contour with occasional protuberances. Furthermore, the analysis of Fig. 2d (enlarged portion of Fig. 2c) reveals some interesting facts: the area within red dotted box (Fig. 2d) appears to be cytoplasm oozing out from an opening created in lipopolysaccharide (LPS) layer (the opening is supposed to be of sufficient depth extending deep into plasma membrane) in thin stream and making a pool of cytoplasm at the vicinity of cells (shown within black dotted box, Fig. 2d). The path of oozing
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Antonie van Leeuwenhoek Fig. 1 Transmission electron microscopic images of untreated (a–b) and noncationized nanoemulsions (NCN) treated P. aeruginosa cells (c–h). The arrows (b) illustrate the presence of smooth cell wall (CW) and plasma membrane (PM) of untreated bacteria. Black squares indicate the presence of non-adhered NCN (c–h). Black arrows (c–e, h) indicate the NCN attached to bacterial cell wall. Red arrows (c–d, g) and blue arrows (c–f, h) show blebs formation and breakage of cell wall at various points, respectively. The exudations of essential cellular contents at vicinity of cells are shown within red square box (d, g–h). The photomicrographs were taken from different grid areas. Scale bar (a– f) 1000 nm; (g) 2000 nm; (h) 500 nm
cytoplasm leading to the pool of cytoplasm is demonstrated by the red curve arrow (Fig. 2d). The cell wall of this area is witnessed by occurrence of numerous CNEs attached on the surface of bacteria (shown by black arrow). Another interesting finding is that the disintegration of cell wall is not taking place in one particular place or point but rather it is occurring simultaneously throughout out the bacterial surface as shown by red dotted double end arrows (Fig. 2e, f). It appears that CNEs have high
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cell-membrane piercing potential and their foremost biocidal mechanism might be fusion and followed by membrane commotion. The kind of killing mechanism relies more on collision frequency between nanometric CNEs and bacterial cells followed by diffusion through the cell membrane driven by a concentration gradient since CNEs is present in high concentration outside the bacterial surface. Similar findings were also reported by Chang et al., (2012). Furthermore, it is also apparent that the thickness of
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Fig. 2 Transmission electron microscopic images of untreated (a–b) and cationized nanoemulsions (CNE) treated P. aeruginosa cells (c–h). In one of the image (b), a cell dividing from middle into two halves owing to the creation of septum was observed (shown by arrow). The un-adhered CNE and adhered CNE are shown within black squares (c–h) and black arrows (c– h) respectively. The bleb formations are shown by red arrows
(c, e, g–h) and breakage of cell wall at various points are shown by red double end arrow (e–f). The exudations of essential cytoplasmic contents are shown within black dotted box (d, f). The path of oozing cytoplasm leading to formation of cytoplasm’s pool is demonstrated by red curve arrow (d) and red dotted box (d). The photomicrographs were taken from different grid areas. Scale bar a–b, g 1000 nm; c, h 500 nm; d–f 200 nm
cell wall gradually decreases due to the constant bombardment of numerous CNEs. The reduced thickness of cell wall further enhances the diffusion
of CNEs as per Fick’s law of diffusion that state that the rate of diffusion is indirectly proportional to the thickness of the membrane.
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Cationized lipidic nanoemulsions possess a positive zeta potential of ?20.9 ± 0.46 mV as reported by us previously (Singh et al. 2015a). TEM images of CNEs treated bacterial cell followed by removal of CNEs post treatment indicated no adherence of CNEs on the bacterial surface (supplemental Fig. S1b, d, f). The finding is in contrary to our previous studies with S. aureus where the CNEs remained adhered to the Gram-positive bacteria (Singh et al. 2014). The present findings remains un-answered and requires further studies by incorporating various amounts of oleylamine and making series of CNEs with different degrees of zeta potential which may give optimal value for more efficient electrostatic attractions between negatively charged bacteria. Furthermore, the process of removing CNEs could be carried out at lesser agitation stress so as to observe the presence of electrostatic attractions between oppositely charged cell wall and CNEs. Nevertheless, mild electrostatic attractions between the bacterial cells and CNEs cannot be ruled out. The bactericidal effect of CNEs is evident from supplemental Fig. S1b, d, f that showed the presence of blebs (blue arrows), exudation of cytoplasmic contents from neighbouring cells giving slimy appearance (shown within red square), and deterioration of LPS layer. It is a well known fact that the LPS layer of Gram-negative bacteria acts as a permeability barrier providing stability to the outer membrane and restrict the entry of any foreign particles that might kill or injure the bacteria. Moreover, LPS also contribute to the bacterial attachment to the surface and biofilm formation, apart from providing bacterial resistance to antimicrobial agents (Sotirova et al. 2009). Therefore, it very much evident that turbulence caused by CNEs to the LPS layer will certainly lead to bacterial lysis causing reduced bacterial resistance. The anti-P. aeruginosa effects of NCNs and CNEs were additionally substantiated by SEM studies. The SEM images of control bacteria showed regular rodshaped morphology with smooth cell wall without any structural defects (Fig. 3a, b). However, the NCNs and CNEs treated cells showed evidence of bactericidal effects as shown in Fig. 3c, d and 3e, f, respectively. The extent of cell damage was more with CNEs treated cells as compared to NCNs treated cells. CNEs treated cells had lost the structural integrity with disintegration of cell wall, and leaving behind fragmented residues of cells (shown within red dotted square of
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Fig. 3f). This might be due to the mild electrostatic interaction between cationized nanoemulsions and anionic P. aeruginosa. The negative charge on bacterial surface is mainly due to anionic biophosphorylated sugar head groups of LPS as well as negatively charged lipids like cardiolipin (Ouberai et al. 2011). The enhanced electrostatic interactions lead to the accumulation of CNEs within membrane followed by destabilization of lipid membrane. To further corroborate the comparative efficiency of antibacterial activities of these nanoemulsions (NCNs and CNEs), atomic force microscopy was performed. The AFM images of control P. aeruginosa cells are depicted in Figs. 4a–d and 5a, b. Figure 4a, b portrays 2-D and 3-D AFM images of untreated cells while Fig. 4c, d depicts the contour profile (CP) and dimensional analysis (DA) of the same bacteria. Similarly, Fig. 5a, b illustrates the 2D and 3D AFM images of untreated bacterial cells from different sectional area. These AFM images illustrate the smooth, homogenous and unbroken surface topology in all directions of bacteria with no signs of cell fragmentation. The root mean square (rms) roughness of untreated bacteria was 1.74 ± 0.07, indicating smooth surface sufficing the DA and CP images. The 2-dimensional atomic force microscopic images of NCNs treated cells are portrayed in Fig. 4e while its 3D construction and contour profile is depicted in Fig. 4f and 4g, respectively. The rms roughness P. aeruginosa cells exposed to NCNs treated were found to increase from 1.74 ± 0.07 (control) to 12.7 ± 2.51 nm, demonstrating increased cell roughness. The enhanced rms roughness can further be corroborated from contour profile (Fig. 4g) that showed numerous fragmented remains of bacteria (shown by arrows). The increased roughness might be because of binding of NCNs with lipopolysaccharide (LPS) layer causing the progressive erosion and thereby exposing more roughed thin peptidoglycan (PDG) layer lying beneath to it. The diminished thickness of LPS layer may further facilitate the passage of copious NCNs invading the PDG layer. The NCN may further destabilize the PDG and cellmembrane and may lead to the release of cytoplasmic content containing genetic materials causing fragmentation of bacteria and cell death. The analysis of Fig. 4e–g further reveals that the overall thickness of the cell was diminished sufficing irreparable loss to bacteria because of exudation of cytoplasmic content.
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Fig. 3 Scanning electron microscopy images of untreated P. aeruginosa cells (a–b), non cationized treated (c–d) and cationized treated (e–f). Area within red dotted square indicates the dead residues of bacterial cell when treated with cationized lipidic emulsions
The finding is in accordance with the TEM studies as discussed earlier. Figure 5c–e portrays the 2D, 3D and CP AFM images of CNEs treated P. aeruginosa. The rms roughness of the CNEs treated P. aeruginosa cells was 22.16 ± 3.14 nm, indicative of extensive cell roughness as compared to control (1.74 ± 0.07 nm) and NCN treated cells (12.7 ± 2.51 nm). The enhanced rms was substantiated by the analysis of contour profile (Fig. 5e), where extensive damage was apparent with cell flattening, and relatively more occurrence of fragments as compared to NCNs treated cells (compare Figs. 4g and 5e). The comparatively enhanced fragmentation with increased rms of CNEs treated cell can be ascribed to the mild electrostatic
attraction between positively charged CNE and negatively charged bacterial surface that renders better anchoring to the LPS layer causing successive damages to PDG and cell membrane causing the exudation of essential cytoplasmic contents. Furthermore, it has been reported that the disruption of LPS layer witnessed by increased surface roughness provides indirect evidence that the protein and/or DNA damage contributes significantly to the cytotoxic effects against P. aeruginosa (Deupree and Schoenfisch 2009). In addition to the nanosized droplets and cationization of lipidic emulsions, the composition of lipidic emulsions that includes blends of fatty acids (caprylic acid, caproic acid, lauric acid etc.,) may further enhance the anti-Pseudomonas activities. Any
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Fig. 4 Atomic force microscopic images of untreated (a–d) and non-cationized nanoemulsions (NCN) treated P. aeruginosa cells (e–g). 2-D (a) and 3-D (b) AFM images of untreated bacteria showed no cell deformation while contour profile (c) and
dimensional analysis (d) demonstrates the smooth surface of bacteria without any cell damages. 2-D (e); 3-D (f) and contour profile (g) images of NCN treated bacterial cells showed bacterial fragmentation (shown by arrows, g) leading to cell lysis
molecule that has to traverse through the LPS and plasma membrane layer of Gram-negative bacteria should have optimum hydrophilic-lipophilic balance,
i.e., it should be amphiphilic in nature (having polar and non-polar groups). This is because the LPS and plasma-membrane layer contains polar moieties
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Fig. 5 Atomic force microscopic images of untreated (a– b) and cationized nanoemulsions (CNE) treated P. aeruginosa cells (c–e). 2-D (a) and 3-D (b) AFM images of untreated bacteria showed no cell deformation. 2-D (c); 3-D (d) and
contour profile (e) of CNE treated bacterial cells showed extensive bacterial fragmentation (shown by arrows, e) as compared to NCN treated bacterial cells
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(ethanolamine) that provides hydrophilicity, and nonpolar moieties (fatty acid chains) that imparts hydrophobicity to the rate-limiting barrier. If the molecule is hydrophobic in nature then it may retain only in non-polar part of LPS and will be unable to move forward. Similarly, if the molecule is extremely hydrophilic than it may get concentrated only in the hydrophilic domains and cannot move further deeper into the layer. In the present study, we have selected CMCM8 which is chemically glyceryl mono caprylate (GMC) having HLB value of 4.7, and labrasol which is chemically caprylocaproyl polyoxyl-8 glycerides (CPG) with HLB value of 12, to prepare NCNs and CNEs. Mixed HLB system incorporated into nanoemulsions can efficiently traverse through the LPS and plasma-membrane causing irreparable cell death. Additionally, CPG have been reported to be potent P-pg inhibitor (Lin et al. 2007). It is noteworthy that a major mechanism of P. aeruginosa resistance is the presence of efflux pump embedded in LPS layer that does not allow the entry of drug (Sotirova et al. 2009). CPG being efflux inhibitor may lead to reduced drugresistant of the bacteria.
damage, which is in agreement with the TEM, SEM, and AFM studies.
Agar diffusion and cytoplasmic release studies
Resazurin is an oxidation–reduction indicator used for the evaluation of cell-growth, particularly in various cytotoxic assays (Sarker et al. 2007). It is a blue (weakly fluorescent) and non-toxic dye that turns to pink, when reduced to resorufin by oxidoreductases within viable cells. The MIC was thus determined visually at lowest concentration when no color changes occurred. The representative 96 well microtiter plate showing MIC of NCN and CNE is illustrated in supplemental Fig. S3. The MIC of NCN and CNE was found at 11.37 and 5.70 mg/mL, respectively, demonstrating enhanced anti-Pseudomonas activity of CNE as compared to NCN. The present finding is in accordance with the literature where the positively charged niosomes resulted in the significant reduction in MIC compared to the noncharged niosomes (Abdelaziz et al. 2015). In an another study effectiveness of cationic nanoemulsions (chitosan-coated nanoemulsion) was demonstrated as therapeutics to intervene E. coli induced peritonitis as well as in sepsis (Jain et al. 2014). Furthermore, in a study antimicrobial nanoemulsions were prepared using thyme oil and were cationized by lauric arginate that showed enhanced anti-microbial activity as compared to non-cationized emulsion (Chang et al. 2015).
The representative zone of inhibition exhibited by CNEs (black dotted circle) and NCNs (red dotted circle) against P. aeruginosa is illustrated in supplemental Fig. S2. The inhibition zone was found to be 14.3 ± 1.15 and 10.3 ± 0.6 mm for CNEs and NCNs, respectively. The enhanced inhibition zone exhibited by CNEs can be attributed to mild to moderate adhesion of cationized nanoemulsion with negatively charged bacteria sufficing the findings of TEM, SEM and AFM. The release of UV-absorbing cytoplasmic content from the P. aeruginosa exposed to CNEs and NCNs is an indicator of changes in membrane permeability of bacteria. Bar graph shown in Fig. 6 indicated significant (p \ 0.05) increase of cytoplasmic material when bacterial cells were treated with NCN (0.234 ± 0.023) and CNE (0.425 ± 0.077) as compared to control (0.024 ± 0.010). The t test applied to the mean absorbance values of CNE and NCN treated cells, revealed significant increase in cytoplasmic content in CNE treated cells as compared to NCN (p \ 0.05) illustrating that the CNEs had caused considerable membrane permeation leading to release of cytoplasmic content causing permanent cell
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Percent viable colonies Percent viable colonies of P. aeruginosa remaining after treatment with NCNs and CNEs were determined in time-dependent manner in specific conditions of nutrient media, temperature and time and the results are portrayed in Fig. 6. The control and untreated bacterial cells showed no decline in percent viable colonies at different time intervals (Fig. 6). When bacterial cells were exposed to NCN and CNE, the viable colonies decreased in time-dependent manner. The rate of decline was found higher in CNE as compared to NCN with no observed viable colonies at 60 min (NCN treated) and 15 min (CNE treated). The finding can be attributed to the enhanced adhesion of CNE with bacterial cell walls as compared to NCN, which is in agreement with the previous findings of zone of inhibition and cell constituent release studies. Determination of minimum inhibitory condition
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Fig. 6 Percent viable colonies of P. aeruginosa remaining when treated with non-cationed nanoemulsion (NCN) and cationized nanoemulsion (CNE) at varied time intervals. Bar graph indicates the release of cytoplasmic content exhibited by absorbencies at 260 nm when P. aeruginosa were treated for 1 h with NCN and CNE
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The findings further support the results of morphological studies (TEM, SEM, and AFM) as well as other in vitro anti-Pseudomonas studies (agar diffusion studies, cell constituent studies and time-dependent killing studies)
Conclusions The present study demonstrates the anti-Pseudomonas activities of cationized and non-cationized nanoemulsions using transmission/scanning electron microscopy and atomic force microscopy. The microscopic studies were corroborated by in vitro studies such as zone of inhibition, cytoplasmic release studies, time-dependent killing studies and MIC studies. The microscopic studies demonstrated the cell-lysis due to the formation of blebs, exudation of essential cellular contents and loss of characteristic contours of the cells. The in vitro anti-Pseudomonas studies further supported the findings of microscopic studies. The enhanced inhibition exhibited by cationized nanoemulsions can be attributed to mild to moderate adhesion of cationized nanoemulsion with negatively charged bacteria. In conclusion, it can be inferred that the delivery issues of antibiotics could be reassessed by using lipids that possess inherent antibacterial properties. This will
reduce the dose of antibiotics because of synergistic and tandem effect of excipients and drug acting together. Acknowledgments Authors acknowledges University Grant Commission (UGC), Government of India for providing the financial assistance vide sanction letter number ‘‘41-1423/2012 (SR), dated 30 July 2012, to Mrs. Neeru Singh. The constructive comments and suggestion of anonymous reviewers is gratefully acknowledged. Conflicts of interest Authors report no conflict of interest. The authors alone are responsible for the contents and writing of the paper. Research involving Human participants and/or animals This article does not contain any studies with human participants or animals performed by any of the authors.
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