- Email: [email protected]
Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem
Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
Contents lists available at ScienceDirect
H O S T E D BY
Journal of Radiation Research and Applied Sciences journal homepage: http://www.elsevier.com/locate/jrras
Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem Marwa M. Abdel-Aziz, Mohamed Yosri*, Basma H. Amin The Regional Center for Mycology and Biotechnology, Al-azhar University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 July 2017 Received in revised form 12 September 2017 Accepted 15 September 2017 Available online xxx
Klebsiella pneumoniae (Kp) a common cause of pneumonia leads to intense lung injury and mortality that are correlated with infective exacerbations. A significant increase in the prevalence of imipenem resistant K. pneumoniae (IRKP) has been observed in common human pathogens. The rapid emergence of IRKP has limited the availability of anti-bacterial treatment options. Silver nano-particles (AgNPs) are one of the well-known antibacterial substances showing such multimode antibacterial action. Therefore, AgNPs are suitable candidates for use in combinations with imipenem in order to improve its antibacterial action. Fifteen fungal species were screened for mycosynthesis of silver nanoparticles (AgNPs), only eight fungal species were found to reduce the silver salt into silver nanoparticles which was characterized by UVevisible spectrophotometric analysis, Energy Dispersive Analysis of X-ray (EDX) and transmission electron microscopy. Consistent with characterized silver nanoparticles, mycosynthesized AgNPs by Verticillium albo-atrum (RCMB 039001) was found the higher in concentration (as detected by UVevisible spectrophotometric analysis) and the least in size (as detected by TEM analysis) so, it was chosen for further studies such as in vitro antibacterial activity against Imipenem resistant K. pneumonia (IRKP), MIC, FIC measurements and in vivo study. In this work a strong synergistic antibacterial effect between AgNPs and imipenem was detected in vitro (with FIC Index 0.07) and in vivo against IRKP strain. These results suggested that sliver nanoparticles have an effective antibacterial action on bacterial count, histopathology as well as protective immune response in an IRKP rat model. © 2017 The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Keywords: Imipenem resistant K. pneumonia Silver nano-particles Verticillium albo-Atrum Animal models Antibacterial action
1. Introduction Klebsiella pneumoniae (Kp) is a Gram-negative, nonmotile, encapsulated, lactose-fermenting, facultative anaerobic, rodshaped bacterium of worldwide importance causing pneumonia associated with high morbidity and mortality (Armstrong, Conn, & Pinner, 1999; Mizgerd, 2006). Pneumonia caused by Kp is characterized by an exacerbated inflammatory response, associated with excessive neutrophil and macrophage infiltration, high production of pro-inflammatory cytokines and severe lung injury (Soares et al., 2006; Zhang, Summer, Bagby, & Nelson, 2000). Although local inflammation is beneficial following pathogen infection by
* Corresponding author. E-mail address: [email protected] (M. Yosri). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications.
preventing pathogen dissemination, non-resolving hyper-inflammation is accompanied by mortality and chronic inflammatory disorders (Medzhitov, 2008). Kp infection is being recognized as a major health threat due to the increasing antibiotic-resistance therefore limiting efficient therapies. Alternatively, strategies to reprogram lung defences and improve immune response to clear bacteria could be effective against pulmonary Kp infection. In this context, probiotics have emerged as a strong potential candidate. (Reid, Jass, Sebulsky, & McCormick, 2003). Klebsiella pneumoniae is one of the pathogens responsible for the majority of hospital infections in the United States (Boucher et al., 2009). Resistance of K. pneumoniae to carbapenem antibiotics has spread to all regions of the world and in some countries carbapenem resistance is present in more than half of the patients treated for K. pneumoniae infections (WHO, 2014). K. pneumoniae is also emerging as an agent of severe community-acquired infection including bacteraemia pneumonia (Lin, Jeng, Chen, & Fung, 2010).
https://doi.org/10.1016/j.jrras.2017.09.002 1687-8507/© 2017 The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
2
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
Imipenem is recommended as a first-line therapy for multi-drug resistant Gram-negative bacteria; however, resistance to imipenem has emerged (Ardanuy et al., 1998; Cao et al., 2000; Gülmez et al., 2008; Normann, Cuzon, & Naas, 2009). The mechanism of imipenem resistance involves the production of specific carbapenemases nech-Snchez, Hernndezas well as the loss of porin proteins (Dome s, Martnez-Martnez, Bened, & Albert, 1999; Hesna et al., 2002; Alle Yang, Guo, & Zhang, 2009). Imipenem resistance caused by the alteration in lipopolysaccharide (LPS) levels has also been reported in Enterobacter aerogenes (Bornet, Davin-Regli, Bosi, Pages, & Bollet, 2000; Leying, Cullmann, & Dick, 1991). In recent years, the application of bio nanotechnology has been investigated as an alternative to chemical and physical ones. Research in bio-nanotechnology has shown to provide reliable, ecofriendly processes for synthesis of noble nanomaterials. Biological synthesis of nanoparticles using various biological systems such as yeast, bacteria, fungi, algae and plant extract have been reported (Yen & Mashitah, 2012). A large number of fungal strains are capable to synthesize silver nanoparticles (AgNPs) extracellularly, among which Fusarium oxysporum (Ahmad et al., 2003), Aspergillus fumigatus (Bhainsa & D'Souza, 2006), Aspergillus niger (Gade et al., 2008), Fusarium semitectum (Basavaraja, Balaji, Lagashetty, Rajasab, & Venkataraman, 2008), Penicillium brevicompactum (Shaligram et al., 2009), Cladosporium cladosporioides (Balaji et al., 2009), and Aspergillus clavatus (Verma, Kharwar, & Gange, 2010) have been previously described. Recently, several studies have indicated that AgNPs may strengthen the antibacterial effects of antibiotics against both susceptible and resistant bacteria, either additively or synergistically. The additive effect was shown in antibiotics of different mode of action against various bacterial strains (Birla et al., 2009; Fayaz et al., 2010; Ghosh et al., 2012; Naqvi et al., 2013; Muhsin & Hachim, 2014). In the present study, we attempted to evaluate antibacterial activity of characterized mycosynthesized AgNPs singly and in combination with imipenem against imipenem resistant -Klebsiella pneumonia. 2. Materials and methods 2.1. Fungal species Alternaria tenuissima (RCMB009006), Aspergillus clavatus (RCMB0020161), Aspergillus oryzae (RCMB002015), Cladosporium herbarum (RCMB027002), Emericella nidulans (RCMB004002), Eurotium repens (RCMB003003), Fusarium equiseti (RCMB008004), Fusarium oxysporum (RCMB008006), Kluyveromyces marxianus (RCMB007004), Penicillium echinulatum (RCMB0010042), Penicillium hordei (RCMB0110056), Rhizoctonia solani (RCMB031004), Rhizopus microsporus (RCMB014004), Trichoderma longibrachiatum (RCMB017007) and Verticillium albo-atrum (RCMB039001) were kindly provided from the Regional Centre for Mycology and Biotechnology.
to Kathiresan, Manivannan, and Nabeel (2009). 2.3. Biosynthesis of AgNPs For biosynthesis of AgNPs, 50-ml of cell filtrate was mixed with 10-ml AgNO3 solution (1 mM) and reaction mixture without AgNO3 was used as control. The prepared solutions were incubated at 28 C for 72 h. All solutions were kept in dark to avoid any photochemical reactions during the experiment. The AgNPs were collected by centrifugation at 10,000 rpm for 10 min twice, and collected for further characterization according to Kathiresan et al. (2009). 2.4. Characterization of AgNPs After 24 h of synthesis, Repeated rinses were performed to remove impurities by centrifugation at 14,000 rpm for 30 min at room temperature. The pellet of AgNPs was re-suspended in 1 ml sterile water. The production of AgNPs in aqueous solution was monitored at the Regional Centre for Mycology and Biotechnology (RCMB) using. i. UVevisible Spectroscopy Analysis: Change in colour of the cell free filtrate incubated with silver nitrate solution was visually observed over a period of time. Absorption measurements were carried out using UVevisible spectrophotometer (Milton-Roy Spectronic 1201). UVeVisible analysis of several weeks old samples was also carried out to check the stability of synthesized AgNPs. ii. Transmission Electron Microscopy (TEM): For TEM analysis, a drop of the solution was placed on the carbon coated copper grids and dried by allowing water to evaporate at room temperature. Electron micrographs were obtained using GEOL GEM-1010 transmission electron microscope at 70 kV. iii. Energy Dispersive Analysis of X-ray (EDX): The presence of elemental silver was confirmed through EDX. The EDX microanalysis was carried out by X-ray micro-analyzer (Oxford 6587 INCA) attached to JEOL JSM-5500 LV scanning electron microscope at 20 kV. The EDX spectrum recorded in the spot profile mode from one of the densely populated silver nanoparticles region on the surface of the film. The nano crystallites were analyzed using Quanta 200 FEG.
2.5. Bacterial strains Imipenem resistant K. pneumoniae clinical strain was kindly provided from culture and collection unit of the Regional Centre for Mycology and Biotechnology with code (RCMB 5621). 2.6. Antibiotic Imipenem was purchased from SIGMA. Imipenem was dissolved in 10 mM phosphate buffer (pH 7.0).
2.2. Fungal biomass preparation All fungi under investigation were grown on malt extract broth at 28 C on a rotary shaker (120 rpm) for 96 h. The biomasses were harvested by filtration using Whatman filter paper No. 1, followed by washing with distilled water to remove any components of the medium. The biomass (25 gm) wet weight was placed in individual flasks containing 100 ml of deionized water and incubated as described above for 24 h. The biomass was filtered, and the cell filtrate was collected and used for biosynthesis of AgNPs according
2.7. Determination of minimum inhibitory concentration (MIC) and fractional inhibitory concentrations (FIC) of imipenem and Agnanoparticles The MICs of imipenem, AgNPs and combined imipenemeAgNPs for imipenem resistant K. pneumoniae RCMB5621 determined by the agar dilution method using MH agar. MIC was defined as the lowest drug concentration that inhibited bacterial growth. To measure the synergistic effect of AgNPs and imipenem against IRKP
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
strains, fractional inhibitory concentrations (FICs) were measured according to the following formula:
MIC of imipenem plus AgNPs MIC of AgNPs plus imipenem þ MIC of imipenem alone MIC of AgNPs alone FIC indices were interpreted as synergistic when values were 0.5, as additive or indifferent when values were >0.5e4.0 and as antagonistic when values were >4.0. 2.8. Animals and treatments Male Wister albino rats weighing 160e180 g were obtained from the Animal House Colony of the National Research Centre of Egypt and divided into five groups (nine rats each) after a week of acclimatization. These were maintained in the animal house of Faculty of Science, Al-Azhar University, where they were put in standard cages with food and water and at optimum conditions included temperature, light and ventilation. The first group was the negative control group (NC) that was injected with saline. All other groups were injected intratracheally with 0.1 ml of imipenem resistant K. pneumoniae RCMB5621 suspension (1 108 CFU/ml) to induce bacterial infection. The second group was the positive control group (PC) that remained untreated. The third and fourth groups were injected orally every day with 0.1 ml of imipenem (Sigma) and 0.1 ml of AgNPs respectively (the dose given was depended on MIC), the dose depended to the weight of the body 40 mg/kg/day and interaction (antibiotic þ AgNPs each/each), respectively. The fifth group was also treated orally daily with combination of imipenem and AgNPs (depended on MIC of AgNPs & imipenem combination). The experimental protocol was approved by the Scientific Research Ethics Committee of the Faculty of Medicine, Al-Azhar University. Animals were sacrificed after 3, 7 and 14 days under light ether anaesthesia to further experiments. 2.9. Colony forming units (CFU) To determine lung microbial burden, lungs were excised, washed and homogenized in sterile water. Lung homogenates were serially diluted in sterile water and then 0.1 of it were plated on HiCrome Klebsiella Selective Agar Base plates in triplicates in10-ml aliquots and incubated at 37 C. K. pneumoniae RCMB5621 colonies were counted one day later, and the number of CFUs was calculated on a per whole lung basis (Shatzkes et al., 2016). 2.10. Histological analysis Lungs were fixed by inflation with 1 ml of 10% neutral buffered formalin then excised and immersed in neutral buffered formalin prior to routine paraffin embedding at thickness of 5 mm. Sections were subsequently stained with hematoxylin and eosin (Bancroft & Stevens, 1996). 2.11. Transmission electron microscope examination Samples of lungs from sacrificed rats were obtained and cut into 1 mm3. Fixation of samples, dehydration, embedding, sectioning, and ultra-sectioning were carried out. Ultra-thin sections were stained according to the methods outlined by Bancroft and Gamble (2008), using uranyl acetate and lead citrate. They were subsequently examined under a TEM (JEOL 1200 EX II, Japan) at the Regional Mycology and Biotechnology Centre, Al-Azhar University, Egypt.
3
3. Results 3.1. Biosynthesis of silver nanoparticles (AgNPs) Fifteen fungal species were screened for their ability to biosynthesis AgNPs, only eight fungal species; namely Aspergillus oryzae (RCMB002015), Cladosporium herbarum (RCMB027002), Emericella nidulans (RCMB004002), Fusarium equiseti (RCMB008004), Fusarium proliferatum (RCMB008006), Penicillium echinulatum (RCMB0010042), Trichoderma longibrachiatum (RCMB017007) and Verticillium albo-atrum (RCMB039001) were found to be able to reduce the silver salt into silver nanoparticles with a gradual change to brown colour, clearly indicating the formation of AgNPs when each was incubated with silver nitrate solution and maintained under dark conditions. 3.2. Characterization of AgNPs 3.2.1. UVevisible spectroscopy analysis The UVevisible spectra of the eight fungal species filtrates treated with the silver nitrate solution showed characteristic surface plasmon absorption at 420 nm with O.D. in the following order Verticillium albo-atrum > Fusarium equiseti > Fusarium proliferatum > Trichoderma longibrachiatum > Cladosporium herbarum > Emericella nidulans > Penicillium echinulatum > Aspergillus oryzae (Fig. 1), depending on the ability of fungi as reducing agents. 3.2.2. Energy Dispersive analysis of X-ray (EDX) EDX gives qualitative as well as quantitative status of elements that may be involved in the formation of AgNPs. EDX micro-analysis is performed by measuring the energy and intensity distribution of X-ray signals generated by a focused electron beam on a specimen. Results presented in Fig. 2A showed the EDX spectrum recorded in the spot-profile mode. From the EDX analysis, it is clear that AgNPs reduced by A. oryzae, C. herbarum, E. nidulans, F. equiseti, F. proliferatum, P. echinulatum, T. longibrachiatum and V. albo-atrum have the weight percentage of silver as 23.2, 62.1, 46.3, 81.3, 79.3, 41.5, 76.2 and 87.3% respectively (Data not shown). 3.2.3. Microscopic characterization by TEM The direct electron microscopic visualization allows measuring the size and shape of the silver nanoparticles formed. The micrograph showed nanoparticles with variable shape, most of them present in spherical in nature. The means size of the particles is ranged from 12.62 to 27.45 nm (Table 1). Majority of the silver nanoparticles were spherical in shape and scattered with only a few of them showing aggregates of varying sizes as observed under TEM (Fig. 2B). 3.3. MICs of imipenem and AgNPs against K. pneumoniae strain and synergistic effect The MICs of imipenem and Ag-nanoparticles for the clinical isolate of K. pneumoniae were evaluated. K. pneumoniae (RCMB 5621) strain was highly resistant to imipenem (IRKP) with MIC 32 mg/ml. The MIC of Ag-nanoparticles exposed to the IRKP isolates was 25 mg/ml. When imipenem was combined with MIC Agnanoparticles, a synergistic effect was observed at a concentration 1 mg/ml with FIC Index 0.07. 3.4. Colony forming unit (CFU) measurements Colony forming units of pulmonary K. pneumoniae (RCMB 5621) strain increased by increasing time intervals in bacterial groups which was in contrary for other groups (Kl þ drug, Kl þ nano and
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
4
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
Fig. 1. UVevisible absorption spectra obtained for silver nanoparticles synthesized by different fungal strains. The peaks at 420 nm correspond to the surface Plasmon resonance of silver nanoparticles.
Fig. 2. EDX spectra of AgNps (A), silver x-ray emission peaks are labeled, TEM micrograph of AgNps biosynthesized by different fungi (B). Scale bar ¼ 100 nm.
Kl þ combination); CFUs were drastically decreased as affected by Ag-nanoparticles and when imipenem was combined with Ag-
nanoparticles, a strong synergistic effect between nanoparticles and imipenem was observed (Fig. 3).
Ag-
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
5
Table 1 Statistical measurements of AgNPs using TEM. Fungal strains
Count
Mean
Minimum
Maximum
Standard Deviation
20.48 15.45 18.59 14.27 14.87 18.09 14.72 8.09
36.14 27.79 24.59 18.22 21.82 26.91 22.42 16.91
4.95 4.24 4.24 1.19 2.15 2.52 2.38 2.52
nm A. oryzae (RCMB002015) C.herbarum (RCMB027002) E.nidulans (RCMB004002) F. equiseti (RCMB008004) F.proliferatum (RCMB008006) P. echinulatum (RCMB0010042) T. longibrachiatum (RCMB017007) V. albo-atrum (RCMB039001)
25 25 25 25 25 25 25 25
27.45 19.90 20.84 15.79 17.16 22.62 18.56 12.62
Fig. 3. Histogram represent K. pneumoniae (RCMB 5621) Cfus after 3,7 and 14 days as affected by different treatments; C: Negative control group without K. pneumoniae injection; Kl, Kl þ drug, Kl þ nano and Kl þ comb: groups injected with K. pneumoniae but without treatment, treated with imipenem, treated with Ag-nanoparticles and treated with a combination of Ag-nanoparticles & imipenem respectively.
3.5. Imipenem and Ag-nanoparticles promotes pulmonary healing during IRKP infection Bacterial infection leads to severe progression of acute lung injury characterized by diffuse alveolar damage with highly apparent bright pink hyaline streaks of fibrin and severe inflammation. We therefore compared lung histology in five groups of control, infected with K. pneumoniae, infected with K. pneumoniae þ imipenem treatment, K. pneumoniae þ AgNPs treatment and infected with K. pneumoniae þ combination treatment in albino rats. Gradual ascending improvement after different time points (3, 7, 14 days) in the lung structure were began by oral treatment of rats using imipenem followed by AgNPs. While maximum improvement using combination of imipenem and AgNPs as shown in (Fig. 4a). Additionally, sections of different groups of lungs obtained and stained with hematoxylin and eosin from different areas and at 40 power. We note the presence of classically activated macrophages after different treatments which represent basic macrophage population and functions in host defence as shown in Fig. 4. To confirm the effect of treatments on bacteria and cell structure transmission electron microscope was used to examine ultrathin modifications in lung structure after 14 days. We note that bacterial cells start to be attacked by lung macrophages, however upon oral treatment of imipenem, bacterial number started to be decreased
with moderate effects in cell structure. Moreover, AgNPs induced lung macrophages to better engulf bacterial cells. While combination of AgNPs and imipenem help lung cells to complete eradication of bacterial infection as shown in Fig. 5. 4. Discussion Fungi are commonly used in the biosynthesis of inorganic NPs in comparison to bacteria because of higher output and their easy handling (Hulkoti & Taranath, 2014; Iravani, 2014). Recovery of NPs from fungi media is very simple by pure water washing (Vahabi, Mansoori, & Karimi, 2011). In contrast, the chemical methods use toxic solvents like 1, 2 hexadecanediol, oleylamine, phenyl ether (Sun et al., 2004), 1-hexadecene, octyl ether, 1-octadecene, 1eicosene, and trioctylamine (Park et al., 2004) to recover NPs. This produces NPs with hydrophobic surfaces that should be converted to hydrophilic by applying extra steps (Moon et al., 2010). In the current study filtrates of eight fungal species showed changes in colour from almost yellow to brown; this is a clear indicator of the formation of silver nanoparticles in the reaction mixture. Formation of dark brown is due to the surface Plasmon resonance property of silver nanoparticles (Hemath, Gaurav, Karthik, & Rao, 2010; Ravishankar & Jamuna, 2011; Sangeetha, Rajeshwari, & Venckatesh, 2012; Soheyla, Barabadi, GharaeiFathabad, & Naghibi, 2013; Yen & Mashitah, 2012). UVevis
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
6
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
Fig. 4. A) Imipenem and Ag-nanoparticles upregulate antibacterial mechanisms during IRKP infection. Albino rats were infected intratracheally with IRKP to establish a pulmonary infection. (A) Sections of five groups (1: control group, 2: Infected with Kl, 3: Kl þ drug, 4:Kl þ nano and 5:Kl þ combination), lungs obtained after 3, 7 and 14 days and were stained with H&E. Representative images from different areas are shown at 20 power. Note the severe progression of acute lung injury characterized by diffuse alveolar damage in the second group, while stages of improvement after several treatments. B): Sections of five groups of lungs after 14 days and were stained with H&E. Representative images from different areas are shown at 40 power. Note the predominance of alternatively activated macrophages (black arrows) in the second group. While presence of both classically activated macrophages (white arrows) and alternatively activated macrophages in the third and fourth group.
spectroscopy was used to record the formation of AgNPs by reduction of AgNO3 by fungi. The results show strong surface Plasmon resonance centred at 420 nm which indicates the formation of silver nanoparticles, control without silver ions showed no change in colour when incubated under the same conditions. The shape and size of the result particles were elucidated with the TEM. Nanoparticles observed are spherical with a small percentage of elongated particles. It is a variation in particle size, and the average sizes were 27.45, 19.90, 20.84, 15.79, 17.16, 22.62, 18.56 and 12.62 nm for Aspergillus oryzae (RCMB002015), Cladosporium herbarum (RCMB027002), Emericella nidulans (RCMB004002), Fusarium equiseti (RCMB008004), Fusarium proliferatum (RCMB008006), Penicillium echinulatum (RCMB0010042), Trichoderma longibrachiatum (RCMB017007) and Verticillium albo-atrum (RCMB039001) respectively. The obtained mycosynthesized
nanoparticles are in the range of size approximately 1e50 nm and few particles are agglomerated as indicated by Narasimha, JanardhanAlzohairy, Khadri, and Mallikarjuna (2013). Mycosynthesized AgNPs using Verticillium albo-atrum (RCMB039001) as mediator were noticed to have the highest optical density at 420 nm among all tested mycosynthesized AgNPs which indicated the highest concentration of AgNPs beside the least size with range 8.09e16.91 nm and those two indicators are very important for bioactivity of AgNPs, so mycosynthesized AgNPs using Verticillium albo-atrum (RCMB039001) is going to be used for further study. The MIC for imipenem was decreased for IRKP strain. These results demonstrate that Ag-nanoparticles were synergistic with imipenem. This finding was consistent with previous reports that a special synergy effect can occur when antibiotics are combined
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
7
Fig. 5. TEM micrographs of (1) normal lung macrophage after 14 days with normal nucleus and normal organelles, (2) lung macrophage after 14 days of bacterial infection with irregular nucleus and predominance of bacterial cells (white arrow), (3) lung macrophage after treatment with imipenem for 14 days with improved nucleus and presence of bacterial cells (white arrow), (4) lung macrophage after treatment with Ag-nanoparticles (black arrow) for 14 days with improved nucleus and presence of bacterial cells inside cells (white arrow) and (5) cured lung cell after treated with combination Ag-nanoparticles and imipenem (after 14 days).
with an agent that antagonizes bacterial resistance mechanisms (Wagner & Ulrich-Merzenich, 2009). AgNPs themselves at concentrations below 3 mg/L do not display a cytotoxic effect to human cells or blood as well as to environmentally important organisms as was proved in earlier studies (Panacek et al., 2009, 2011; Kvitek et al., 2009;; Krajewski et al., 2013). Our studies demonstrate that AgNPs can interfere with host responses to pulmonary IRKP infection. Combination of AgNPs and imipenem against IRKP pulmonary challenge results in microbial clearance. To assess the activation state of lung cells, we examined effect of imipenem, AgNPs and their combination in tissue of lung after bacterial infection at several time points. Our results suggest that different treatments induce (M1) classically activated macrophages against alternatively activated macrophages (M2). Leopold and Wormley (2014) demonstrated the critical role classically and alternatively activated macrophages during respiratory fungal infections and how the outcome of infection can be determined by the macrophages ability to polarize in a way that supports resolution and not exacerbation of disease. In summary, this study has demonstrated the effects of AgNPs in combination of imipenem to extended to it antibacterial effects against IRKP infection suggesting that future therapeutic strategies with feasible way to improve management of Klebsiella pneumonia infections. References Ahmad, A., Mukherjee, P., Senapati, S., Mandal, D., Khan, M. I., Kumar, R., et al. (2003). Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloids and Surfaces B: Biointerfaces, 28(4), 313e318. s, S., Bened, V. J., & MartArdanuy, C., Lienares, J., Domnguez, M. A., Hernndez-Alle nez-Martnez, L. (1998). Outer membrane profiles of clonally related Klebsiella pneumoniae isolates from clinical samples and activities of cephalosporins and carbapenems. Antimicrobial Agents and Chemotherapy, 42, 1636e1640. Armstrong, G. L., Conn, L. A., & Pinner, R. W. (1999). Trends in infectious disease mortality in the United States during the 20th century. JAMA, 281, 61e66. Balaji, D. S., Basavaraja, S., Deshpande, R., Mahesh, D. B., Prabhakar, B. K., & Venkataraman, A. (2009). Extracellular biosynthesis of functionalized silver nanoparticles by strains of Cladosporium cladosporioides fungus. Colloids and Surfaces B: Biointerfaces, 68(1), 88e92. Bancroft, J. D., & Gamble, M. (2008). Theory and practice of histological techniques (6th ed.). Philadelphia, PA: Churchill Livingstone. Bancroft, J. D., & Stevens, A. L. (1996). Theory and practice of histological techniques (4th ed.). Edinburgh: Churchill Living stone. Basavaraja, S., Balaji, S. D., Lagashetty, A., Rajasab, A. H., & Venkataraman, A. (2008).
Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium semitectum. Materials Research Bulletin, 43(5), 1164e1170. Bhainsa, K. C., & D'Souza, S. F. (2006). Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigatus. Colloids and Surfaces B: Biointerfaces, 47(2), 160e164. Birla, S. S., Tiwari, V. V., Gade, A. K., Ingle, A. P., Yadav, A. P., & Rai, M. K. (2009). Fabrication of silver nanoparticles by phoma glomerata and its combined effect against Escherichia coli, pseudomonas aeruginosa and staphylococcus aureus. Letters in Applied Microbiology, 48, 173e179. Bornet, C., Davin-Regli, A., Bosi, C., Pages, J. M., & Bollet, C. (2000). Imipenem resistance of Enterobacter aerogenes mediated by outer membrane permeability. Journal of Clinical Microbiology, 38, 1048e1052. Boucher, H. W., Talbot, G. H., Bradley, J. S., Edwards, J. E., Gilbert, D., & Rice, L. B. (2009). Bad bugs, no drugs: no ESKAPE! An update from the infectious diseases society of America. Clinical Infectious Diseases, 48, 1e12. Cao, V. T., Arlet, G., Ericsson, B. M., Tammelin, A., Courvalin, P., & Lambert, T. (2000). Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration. Journal of Antimicrobial Chemotherapy, 46, 895e900. nech-Snchez, A., Hernndez-Alle s, S., Martnez-Martnez, L., Bened, V. J., & Dome Albert, S. (1999). Identification and characterization of a new porin gene of Klebsiella pneumoniae: Its role in beta-lactam antibiotic resistance. Journal of Bacteriology, 181, 2726e2732. Fayaz, A. M., Balaji, K., Girilal, M., Yadav, R., Kalaichelvan, P. T., & Venketesan, R. (2010). Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: A study against gram-positive and gram-negative bacteria. Journal of Nanomedicine & Nanotechnology, 6, 103e109. n, N., & Rai, M. K. (2008). Gade, A. K., Bonde, P., Ingle, A. P., Marcato, P. D., Dura Exploitation of Aspergillus niger for synthesis of silver nanoparticles. Journal of Biobased Materials and Bioenergy, 2(3), 243e247. Ghosh, S., Patil, S., Ahire, M., Kitture, R., Kale, S., Pardesi, K., et al. (2012). Synthesis of silver nanoparticles using Dioscorea bulbifera tuber extract and evaluation of its synergistic potential in combination with antimicrobial agents. International Journal of Nanomedicine, 7, 483e496. Gülmez, D., Woodford, N., Palepou, M. F., Mushtaq, S., Metan, G., Yakupogullari, Y., et al. (2008). Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates from Turkey with OXA-48-like carbapenemases and outer membrane protein loss. International Journal of Antimicrobial Agents, 31, 523e526. Hemath, N. K. S., Gaurav, K., Karthik, L., & Rao, K. V. (2010). Extracellular biosynthesis of silver nanoparticles using the filamentous fungus Penicillium sp. Archives of Applied Science Research, 2, 161e167. Hesna, Y., Anderson, G. J., Biddle, J. W., Steward, C. D., Rasheed, J. K., Valera, L. L., et al. (2002). Carbapenem resistance in a clinical isolate of Enterobacter aerogenes is associated with decreased expression of OmpF and OmpC porin analogs. Antimicrobial Agents and Chemotherapy, 46, 3817e3822. Hulkoti, N. I., & Taranath, T. C. (2014). Biosynthesis of nanoparticles using microbes e a review. Colloids and Surfaces B: Biointerfaces, 121, 474e483. Iravani, S. (2014). Bacteria in nanoparticle synthesis: Current status and future prospects. International Scholarly Research Notices, 1e18. Kathiresan, K., Manivannan, M. A., & Nabeel, B. (2009). Studies on silver nanoparticles synthesized by a marine fungus, Penicillium fellutanum isolated from coastal mangrove sediment. Colloids and Surfaces B: Biointerfaces, 71, 133e137. Krajewski, S., Prucek, R., Panacek, A., Avci-Adali, M., Nolte, A., Straub, A., et al. (2013). Hemocompatibility evaluation of different silver nanoparticle concentrations
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
8
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
employing a modified chandler-loop in vitro assay on human blood. Acta Biomaterialia, 9, 7460e7468. Kvitek, L., Vanickova, M., Panacek, A., Soukupova, J., Dittrich, M., Valentova, E., et al. (2009). Initial study on the toxicity of silver nanoparticles (nps) against paramecium caudatum. Journal of Physical Chemistry C, 113, 4296e4300. Leopold, W. C. M., & Wormley, F. L. (2014). Classical versus alternative macrophage activation: The ying and the Yang in host defense against pulmonary fungal infections. Mucosal Immunology, 7, 1023e1035. Leying, H., Cullmann, W., & Dick, W. (1991). Carbapenem resistance in Enterobacter aerogenes is due to lipopolysaccharide alterations. Chemotherapy, 37, 106e113. Lin, Y. T., Jeng, Y. Y., Chen, T. L., & Fung, C. P. (2010). Bacteremic community-acquired pneumonia due to Klebsiella pneumoniae: Clinical and microbiological characteristics in Taiwan. BMC Infectious Diseases, 10, 307. Medzhitov, R. (2008). Origin and physiological roles of inflammation. Nature, 454, 428e835. Mizgerd, J. P. (2006). Lung infection public health priority. PLoS Medicine, 3, 76. Moon, J. W., Rawn, C. J., Rondinone, A. J., Love, L. J., Roh, Y., Everett, S. M., et al. (2010). Large-scale production of magnetic nanoparticles using bacterial fermentation. Journal of Industrial Microbiology and Biotechnology, 37, 1023e1031. Muhsin, T. M., & Hachim, A. K. (2014). Mycosynthesis and characterization of silver nanoparticles and their activity against some human pathogenic bacteria. World Journal of Microbiology and Biotechnology, 30, 2081e2090. Naqvi, S. Z. H., Kiran, U., Ali, M. I., Jamal, A., Hameed, A., Ahmed, S., et al. (2013). Combined efficacy of biologically synthesized silver nanoparticles and different antibiotics against multidrug-resistant bacteria. International Journal of Nanomedicine, 8, 187e195. Narasimha, G., Janardhan Alzohairy, M., Khadri, H., & Mallikarjuna, K. (2013). Extracellular syenthesis, characterization and antibacterial activity of silver nanoparticles by Actinomycetes isolative. International Journal of Nanodimens, 4, 77e83. Normann, P., Cuzon, G., & Naas, T. (2009). The real threat of Klebsiella pneumoniae carbapenemase producing bacteria. Lancet Infectious Diseases, 9, 228e236. Panacek, A., Kolar, M., Vecerova, R., Prucek, R., Soukupova, J., Krystof, V., et al. (2009). Antifungal activity of silver nanoparticles against candida spp. Biomaterials, 30, 6333e6340. Panacek, A., Prucek, R., Safarova, D., Dittrich, M., Richtrova, J., Benickova, K., et al. (2011). Acute and chronic toxicity effects of silver nanoparticles (nps) on drosophila melanogaster. Environmental Science and Technology, 45, 4974e4979. Park, J., An, K., Hwang, Y., Park, J. G., Noh, H. J., Kim, J. Y., et al. (2004). Ultra-largescale syntheses of monodisperse nanocrystals. Nature Materials, 3, 891e895. Ravishankar, R. V., & Jamuna, B. A. (2011). Nanoparticles and their potential
application as antimicrobials. Communicating Current Research Technological Advances, 197e209. Reid, G., Jass, J., Sebulsky, M. T., & McCormick, J. K. (2003). Potential uses of probiotics in clinical practice. Clinical Microbiology Reviews, 16, 658e672. Sangeetha, G., Rajeshwari, S., & Venckatesh, R. (2012). Green synthesized ZnO nanoparticles against bacterial and fungal pathogens. Progress in Natural Science: Materials International, 6, 693e700. Shaligram, N. S., Bule, M., Bhambure, R., Singhal, R. S., Singh, S. K., et al. (2009). Biosynthesis of silver nanoparticles using aqueous extract from the compactin producing fungal strain. Process Biochemistry, 44(8), 939e943. Shatzkes, K., Singleton, E., Tang, C., Zuena, M., Shukla, S., Gupta, S., et al. (2016). Predatory bacteria attenuate Klebsiella pneumoniae burden in rat lungs. mBio, 7(6). e01847e16. Soares, A. C., Souza, D. G., Pinho, V., Vieira, A. T., Nicoli, J. R., & Cunha, F. Q. (2006). Dual function of the long pentraxin PTX3 in resistance against pulmonary infection with Klebsiella pneumoniae in transgenic mice. Microbes and Infection, 8, 1321e1329. Soheyla, H., Barabadi, H., Gharaei-Fathabad, E., & Naghibi, F. (2013). Green syntheesis of silver nanoparticles induced by the fungus Penicillium citrinum. Tropical Journal of Pharmaceutical Research, 12, 7e11. Sun, S., Zeng, H., Robinson, D. B., Raoux, S., Rice, P. M., Wang, S. X., et al. (2004). MonodisperseMFe2O4 (M ¼ Fe, Co, Mn) nanoparticles. Journal of the American Chemical Society, 126, 273e279. Vahabi, K., Mansoori, G. A., & Karimi, S. (2011). Biosynthesis of silver nanoparticles by fungus Trichoderma reesei (A route for large-scale production of AgNPs). Insciences Journal, 1(1), 65e79. Verma, V. C., Kharwar, R. N., & Gange, A. C. (2010). Biosynthesis of antimicrobial silver nanoparticles by the endophytic fungus Aspergillus clavatus. Nanomedicine (Lond)., 5(1), 33e40. Wagner, H., & Ulrich-Merzenich, G. (2009). Synergy research: Approaching a new generation of phytopharmaceuticals. Phytomedicine, 16, 97e110. WHO. (2014). Antimicrobial resistance: Global report on surveillance. Yang, D., Guo, Y., & Zhang, Z. (2009). Combined porin loss and extended spectrumlactamase production is associated with an increasing imipenem minimal inhibitory concentration in clinical Klebsiella pneumoniae strains. Current Microbiology, 58, 366e370. Yen, S. C., & Mashitah, M. D. (2012). Characterization of Ag nanoparticles produced by white erot fungi and its in vitro antimicrobial activities. The International Arabic Journal of Antimicrobial Agents, 2, 1e8. Zhang, P., Summer, W. R., Bagby, G. J., & Nelson, S. (2000). Innate immunity and pulmonary host defence. Immunological Reviews, 173, 39e51.
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
Contents lists available at ScienceDirect
H O S T E D BY
Journal of Radiation Research and Applied Sciences journal homepage: http://www.elsevier.com/locate/jrras
Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem Marwa M. Abdel-Aziz, Mohamed Yosri*, Basma H. Amin The Regional Center for Mycology and Biotechnology, Al-azhar University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 July 2017 Received in revised form 12 September 2017 Accepted 15 September 2017 Available online xxx
Klebsiella pneumoniae (Kp) a common cause of pneumonia leads to intense lung injury and mortality that are correlated with infective exacerbations. A significant increase in the prevalence of imipenem resistant K. pneumoniae (IRKP) has been observed in common human pathogens. The rapid emergence of IRKP has limited the availability of anti-bacterial treatment options. Silver nano-particles (AgNPs) are one of the well-known antibacterial substances showing such multimode antibacterial action. Therefore, AgNPs are suitable candidates for use in combinations with imipenem in order to improve its antibacterial action. Fifteen fungal species were screened for mycosynthesis of silver nanoparticles (AgNPs), only eight fungal species were found to reduce the silver salt into silver nanoparticles which was characterized by UVevisible spectrophotometric analysis, Energy Dispersive Analysis of X-ray (EDX) and transmission electron microscopy. Consistent with characterized silver nanoparticles, mycosynthesized AgNPs by Verticillium albo-atrum (RCMB 039001) was found the higher in concentration (as detected by UVevisible spectrophotometric analysis) and the least in size (as detected by TEM analysis) so, it was chosen for further studies such as in vitro antibacterial activity against Imipenem resistant K. pneumonia (IRKP), MIC, FIC measurements and in vivo study. In this work a strong synergistic antibacterial effect between AgNPs and imipenem was detected in vitro (with FIC Index 0.07) and in vivo against IRKP strain. These results suggested that sliver nanoparticles have an effective antibacterial action on bacterial count, histopathology as well as protective immune response in an IRKP rat model. © 2017 The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Keywords: Imipenem resistant K. pneumonia Silver nano-particles Verticillium albo-Atrum Animal models Antibacterial action
1. Introduction Klebsiella pneumoniae (Kp) is a Gram-negative, nonmotile, encapsulated, lactose-fermenting, facultative anaerobic, rodshaped bacterium of worldwide importance causing pneumonia associated with high morbidity and mortality (Armstrong, Conn, & Pinner, 1999; Mizgerd, 2006). Pneumonia caused by Kp is characterized by an exacerbated inflammatory response, associated with excessive neutrophil and macrophage infiltration, high production of pro-inflammatory cytokines and severe lung injury (Soares et al., 2006; Zhang, Summer, Bagby, & Nelson, 2000). Although local inflammation is beneficial following pathogen infection by
* Corresponding author. E-mail address: [email protected] (M. Yosri). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications.
preventing pathogen dissemination, non-resolving hyper-inflammation is accompanied by mortality and chronic inflammatory disorders (Medzhitov, 2008). Kp infection is being recognized as a major health threat due to the increasing antibiotic-resistance therefore limiting efficient therapies. Alternatively, strategies to reprogram lung defences and improve immune response to clear bacteria could be effective against pulmonary Kp infection. In this context, probiotics have emerged as a strong potential candidate. (Reid, Jass, Sebulsky, & McCormick, 2003). Klebsiella pneumoniae is one of the pathogens responsible for the majority of hospital infections in the United States (Boucher et al., 2009). Resistance of K. pneumoniae to carbapenem antibiotics has spread to all regions of the world and in some countries carbapenem resistance is present in more than half of the patients treated for K. pneumoniae infections (WHO, 2014). K. pneumoniae is also emerging as an agent of severe community-acquired infection including bacteraemia pneumonia (Lin, Jeng, Chen, & Fung, 2010).
https://doi.org/10.1016/j.jrras.2017.09.002 1687-8507/© 2017 The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
2
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
Imipenem is recommended as a first-line therapy for multi-drug resistant Gram-negative bacteria; however, resistance to imipenem has emerged (Ardanuy et al., 1998; Cao et al., 2000; Gülmez et al., 2008; Normann, Cuzon, & Naas, 2009). The mechanism of imipenem resistance involves the production of specific carbapenemases nech-Snchez, Hernndezas well as the loss of porin proteins (Dome s, Martnez-Martnez, Bened, & Albert, 1999; Hesna et al., 2002; Alle Yang, Guo, & Zhang, 2009). Imipenem resistance caused by the alteration in lipopolysaccharide (LPS) levels has also been reported in Enterobacter aerogenes (Bornet, Davin-Regli, Bosi, Pages, & Bollet, 2000; Leying, Cullmann, & Dick, 1991). In recent years, the application of bio nanotechnology has been investigated as an alternative to chemical and physical ones. Research in bio-nanotechnology has shown to provide reliable, ecofriendly processes for synthesis of noble nanomaterials. Biological synthesis of nanoparticles using various biological systems such as yeast, bacteria, fungi, algae and plant extract have been reported (Yen & Mashitah, 2012). A large number of fungal strains are capable to synthesize silver nanoparticles (AgNPs) extracellularly, among which Fusarium oxysporum (Ahmad et al., 2003), Aspergillus fumigatus (Bhainsa & D'Souza, 2006), Aspergillus niger (Gade et al., 2008), Fusarium semitectum (Basavaraja, Balaji, Lagashetty, Rajasab, & Venkataraman, 2008), Penicillium brevicompactum (Shaligram et al., 2009), Cladosporium cladosporioides (Balaji et al., 2009), and Aspergillus clavatus (Verma, Kharwar, & Gange, 2010) have been previously described. Recently, several studies have indicated that AgNPs may strengthen the antibacterial effects of antibiotics against both susceptible and resistant bacteria, either additively or synergistically. The additive effect was shown in antibiotics of different mode of action against various bacterial strains (Birla et al., 2009; Fayaz et al., 2010; Ghosh et al., 2012; Naqvi et al., 2013; Muhsin & Hachim, 2014). In the present study, we attempted to evaluate antibacterial activity of characterized mycosynthesized AgNPs singly and in combination with imipenem against imipenem resistant -Klebsiella pneumonia. 2. Materials and methods 2.1. Fungal species Alternaria tenuissima (RCMB009006), Aspergillus clavatus (RCMB0020161), Aspergillus oryzae (RCMB002015), Cladosporium herbarum (RCMB027002), Emericella nidulans (RCMB004002), Eurotium repens (RCMB003003), Fusarium equiseti (RCMB008004), Fusarium oxysporum (RCMB008006), Kluyveromyces marxianus (RCMB007004), Penicillium echinulatum (RCMB0010042), Penicillium hordei (RCMB0110056), Rhizoctonia solani (RCMB031004), Rhizopus microsporus (RCMB014004), Trichoderma longibrachiatum (RCMB017007) and Verticillium albo-atrum (RCMB039001) were kindly provided from the Regional Centre for Mycology and Biotechnology.
to Kathiresan, Manivannan, and Nabeel (2009). 2.3. Biosynthesis of AgNPs For biosynthesis of AgNPs, 50-ml of cell filtrate was mixed with 10-ml AgNO3 solution (1 mM) and reaction mixture without AgNO3 was used as control. The prepared solutions were incubated at 28 C for 72 h. All solutions were kept in dark to avoid any photochemical reactions during the experiment. The AgNPs were collected by centrifugation at 10,000 rpm for 10 min twice, and collected for further characterization according to Kathiresan et al. (2009). 2.4. Characterization of AgNPs After 24 h of synthesis, Repeated rinses were performed to remove impurities by centrifugation at 14,000 rpm for 30 min at room temperature. The pellet of AgNPs was re-suspended in 1 ml sterile water. The production of AgNPs in aqueous solution was monitored at the Regional Centre for Mycology and Biotechnology (RCMB) using. i. UVevisible Spectroscopy Analysis: Change in colour of the cell free filtrate incubated with silver nitrate solution was visually observed over a period of time. Absorption measurements were carried out using UVevisible spectrophotometer (Milton-Roy Spectronic 1201). UVeVisible analysis of several weeks old samples was also carried out to check the stability of synthesized AgNPs. ii. Transmission Electron Microscopy (TEM): For TEM analysis, a drop of the solution was placed on the carbon coated copper grids and dried by allowing water to evaporate at room temperature. Electron micrographs were obtained using GEOL GEM-1010 transmission electron microscope at 70 kV. iii. Energy Dispersive Analysis of X-ray (EDX): The presence of elemental silver was confirmed through EDX. The EDX microanalysis was carried out by X-ray micro-analyzer (Oxford 6587 INCA) attached to JEOL JSM-5500 LV scanning electron microscope at 20 kV. The EDX spectrum recorded in the spot profile mode from one of the densely populated silver nanoparticles region on the surface of the film. The nano crystallites were analyzed using Quanta 200 FEG.
2.5. Bacterial strains Imipenem resistant K. pneumoniae clinical strain was kindly provided from culture and collection unit of the Regional Centre for Mycology and Biotechnology with code (RCMB 5621). 2.6. Antibiotic Imipenem was purchased from SIGMA. Imipenem was dissolved in 10 mM phosphate buffer (pH 7.0).
2.2. Fungal biomass preparation All fungi under investigation were grown on malt extract broth at 28 C on a rotary shaker (120 rpm) for 96 h. The biomasses were harvested by filtration using Whatman filter paper No. 1, followed by washing with distilled water to remove any components of the medium. The biomass (25 gm) wet weight was placed in individual flasks containing 100 ml of deionized water and incubated as described above for 24 h. The biomass was filtered, and the cell filtrate was collected and used for biosynthesis of AgNPs according
2.7. Determination of minimum inhibitory concentration (MIC) and fractional inhibitory concentrations (FIC) of imipenem and Agnanoparticles The MICs of imipenem, AgNPs and combined imipenemeAgNPs for imipenem resistant K. pneumoniae RCMB5621 determined by the agar dilution method using MH agar. MIC was defined as the lowest drug concentration that inhibited bacterial growth. To measure the synergistic effect of AgNPs and imipenem against IRKP
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
strains, fractional inhibitory concentrations (FICs) were measured according to the following formula:
MIC of imipenem plus AgNPs MIC of AgNPs plus imipenem þ MIC of imipenem alone MIC of AgNPs alone FIC indices were interpreted as synergistic when values were 0.5, as additive or indifferent when values were >0.5e4.0 and as antagonistic when values were >4.0. 2.8. Animals and treatments Male Wister albino rats weighing 160e180 g were obtained from the Animal House Colony of the National Research Centre of Egypt and divided into five groups (nine rats each) after a week of acclimatization. These were maintained in the animal house of Faculty of Science, Al-Azhar University, where they were put in standard cages with food and water and at optimum conditions included temperature, light and ventilation. The first group was the negative control group (NC) that was injected with saline. All other groups were injected intratracheally with 0.1 ml of imipenem resistant K. pneumoniae RCMB5621 suspension (1 108 CFU/ml) to induce bacterial infection. The second group was the positive control group (PC) that remained untreated. The third and fourth groups were injected orally every day with 0.1 ml of imipenem (Sigma) and 0.1 ml of AgNPs respectively (the dose given was depended on MIC), the dose depended to the weight of the body 40 mg/kg/day and interaction (antibiotic þ AgNPs each/each), respectively. The fifth group was also treated orally daily with combination of imipenem and AgNPs (depended on MIC of AgNPs & imipenem combination). The experimental protocol was approved by the Scientific Research Ethics Committee of the Faculty of Medicine, Al-Azhar University. Animals were sacrificed after 3, 7 and 14 days under light ether anaesthesia to further experiments. 2.9. Colony forming units (CFU) To determine lung microbial burden, lungs were excised, washed and homogenized in sterile water. Lung homogenates were serially diluted in sterile water and then 0.1 of it were plated on HiCrome Klebsiella Selective Agar Base plates in triplicates in10-ml aliquots and incubated at 37 C. K. pneumoniae RCMB5621 colonies were counted one day later, and the number of CFUs was calculated on a per whole lung basis (Shatzkes et al., 2016). 2.10. Histological analysis Lungs were fixed by inflation with 1 ml of 10% neutral buffered formalin then excised and immersed in neutral buffered formalin prior to routine paraffin embedding at thickness of 5 mm. Sections were subsequently stained with hematoxylin and eosin (Bancroft & Stevens, 1996). 2.11. Transmission electron microscope examination Samples of lungs from sacrificed rats were obtained and cut into 1 mm3. Fixation of samples, dehydration, embedding, sectioning, and ultra-sectioning were carried out. Ultra-thin sections were stained according to the methods outlined by Bancroft and Gamble (2008), using uranyl acetate and lead citrate. They were subsequently examined under a TEM (JEOL 1200 EX II, Japan) at the Regional Mycology and Biotechnology Centre, Al-Azhar University, Egypt.
3
3. Results 3.1. Biosynthesis of silver nanoparticles (AgNPs) Fifteen fungal species were screened for their ability to biosynthesis AgNPs, only eight fungal species; namely Aspergillus oryzae (RCMB002015), Cladosporium herbarum (RCMB027002), Emericella nidulans (RCMB004002), Fusarium equiseti (RCMB008004), Fusarium proliferatum (RCMB008006), Penicillium echinulatum (RCMB0010042), Trichoderma longibrachiatum (RCMB017007) and Verticillium albo-atrum (RCMB039001) were found to be able to reduce the silver salt into silver nanoparticles with a gradual change to brown colour, clearly indicating the formation of AgNPs when each was incubated with silver nitrate solution and maintained under dark conditions. 3.2. Characterization of AgNPs 3.2.1. UVevisible spectroscopy analysis The UVevisible spectra of the eight fungal species filtrates treated with the silver nitrate solution showed characteristic surface plasmon absorption at 420 nm with O.D. in the following order Verticillium albo-atrum > Fusarium equiseti > Fusarium proliferatum > Trichoderma longibrachiatum > Cladosporium herbarum > Emericella nidulans > Penicillium echinulatum > Aspergillus oryzae (Fig. 1), depending on the ability of fungi as reducing agents. 3.2.2. Energy Dispersive analysis of X-ray (EDX) EDX gives qualitative as well as quantitative status of elements that may be involved in the formation of AgNPs. EDX micro-analysis is performed by measuring the energy and intensity distribution of X-ray signals generated by a focused electron beam on a specimen. Results presented in Fig. 2A showed the EDX spectrum recorded in the spot-profile mode. From the EDX analysis, it is clear that AgNPs reduced by A. oryzae, C. herbarum, E. nidulans, F. equiseti, F. proliferatum, P. echinulatum, T. longibrachiatum and V. albo-atrum have the weight percentage of silver as 23.2, 62.1, 46.3, 81.3, 79.3, 41.5, 76.2 and 87.3% respectively (Data not shown). 3.2.3. Microscopic characterization by TEM The direct electron microscopic visualization allows measuring the size and shape of the silver nanoparticles formed. The micrograph showed nanoparticles with variable shape, most of them present in spherical in nature. The means size of the particles is ranged from 12.62 to 27.45 nm (Table 1). Majority of the silver nanoparticles were spherical in shape and scattered with only a few of them showing aggregates of varying sizes as observed under TEM (Fig. 2B). 3.3. MICs of imipenem and AgNPs against K. pneumoniae strain and synergistic effect The MICs of imipenem and Ag-nanoparticles for the clinical isolate of K. pneumoniae were evaluated. K. pneumoniae (RCMB 5621) strain was highly resistant to imipenem (IRKP) with MIC 32 mg/ml. The MIC of Ag-nanoparticles exposed to the IRKP isolates was 25 mg/ml. When imipenem was combined with MIC Agnanoparticles, a synergistic effect was observed at a concentration 1 mg/ml with FIC Index 0.07. 3.4. Colony forming unit (CFU) measurements Colony forming units of pulmonary K. pneumoniae (RCMB 5621) strain increased by increasing time intervals in bacterial groups which was in contrary for other groups (Kl þ drug, Kl þ nano and
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
4
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
Fig. 1. UVevisible absorption spectra obtained for silver nanoparticles synthesized by different fungal strains. The peaks at 420 nm correspond to the surface Plasmon resonance of silver nanoparticles.
Fig. 2. EDX spectra of AgNps (A), silver x-ray emission peaks are labeled, TEM micrograph of AgNps biosynthesized by different fungi (B). Scale bar ¼ 100 nm.
Kl þ combination); CFUs were drastically decreased as affected by Ag-nanoparticles and when imipenem was combined with Ag-
nanoparticles, a strong synergistic effect between nanoparticles and imipenem was observed (Fig. 3).
Ag-
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
5
Table 1 Statistical measurements of AgNPs using TEM. Fungal strains
Count
Mean
Minimum
Maximum
Standard Deviation
20.48 15.45 18.59 14.27 14.87 18.09 14.72 8.09
36.14 27.79 24.59 18.22 21.82 26.91 22.42 16.91
4.95 4.24 4.24 1.19 2.15 2.52 2.38 2.52
nm A. oryzae (RCMB002015) C.herbarum (RCMB027002) E.nidulans (RCMB004002) F. equiseti (RCMB008004) F.proliferatum (RCMB008006) P. echinulatum (RCMB0010042) T. longibrachiatum (RCMB017007) V. albo-atrum (RCMB039001)
25 25 25 25 25 25 25 25
27.45 19.90 20.84 15.79 17.16 22.62 18.56 12.62
Fig. 3. Histogram represent K. pneumoniae (RCMB 5621) Cfus after 3,7 and 14 days as affected by different treatments; C: Negative control group without K. pneumoniae injection; Kl, Kl þ drug, Kl þ nano and Kl þ comb: groups injected with K. pneumoniae but without treatment, treated with imipenem, treated with Ag-nanoparticles and treated with a combination of Ag-nanoparticles & imipenem respectively.
3.5. Imipenem and Ag-nanoparticles promotes pulmonary healing during IRKP infection Bacterial infection leads to severe progression of acute lung injury characterized by diffuse alveolar damage with highly apparent bright pink hyaline streaks of fibrin and severe inflammation. We therefore compared lung histology in five groups of control, infected with K. pneumoniae, infected with K. pneumoniae þ imipenem treatment, K. pneumoniae þ AgNPs treatment and infected with K. pneumoniae þ combination treatment in albino rats. Gradual ascending improvement after different time points (3, 7, 14 days) in the lung structure were began by oral treatment of rats using imipenem followed by AgNPs. While maximum improvement using combination of imipenem and AgNPs as shown in (Fig. 4a). Additionally, sections of different groups of lungs obtained and stained with hematoxylin and eosin from different areas and at 40 power. We note the presence of classically activated macrophages after different treatments which represent basic macrophage population and functions in host defence as shown in Fig. 4. To confirm the effect of treatments on bacteria and cell structure transmission electron microscope was used to examine ultrathin modifications in lung structure after 14 days. We note that bacterial cells start to be attacked by lung macrophages, however upon oral treatment of imipenem, bacterial number started to be decreased
with moderate effects in cell structure. Moreover, AgNPs induced lung macrophages to better engulf bacterial cells. While combination of AgNPs and imipenem help lung cells to complete eradication of bacterial infection as shown in Fig. 5. 4. Discussion Fungi are commonly used in the biosynthesis of inorganic NPs in comparison to bacteria because of higher output and their easy handling (Hulkoti & Taranath, 2014; Iravani, 2014). Recovery of NPs from fungi media is very simple by pure water washing (Vahabi, Mansoori, & Karimi, 2011). In contrast, the chemical methods use toxic solvents like 1, 2 hexadecanediol, oleylamine, phenyl ether (Sun et al., 2004), 1-hexadecene, octyl ether, 1-octadecene, 1eicosene, and trioctylamine (Park et al., 2004) to recover NPs. This produces NPs with hydrophobic surfaces that should be converted to hydrophilic by applying extra steps (Moon et al., 2010). In the current study filtrates of eight fungal species showed changes in colour from almost yellow to brown; this is a clear indicator of the formation of silver nanoparticles in the reaction mixture. Formation of dark brown is due to the surface Plasmon resonance property of silver nanoparticles (Hemath, Gaurav, Karthik, & Rao, 2010; Ravishankar & Jamuna, 2011; Sangeetha, Rajeshwari, & Venckatesh, 2012; Soheyla, Barabadi, GharaeiFathabad, & Naghibi, 2013; Yen & Mashitah, 2012). UVevis
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
6
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
Fig. 4. A) Imipenem and Ag-nanoparticles upregulate antibacterial mechanisms during IRKP infection. Albino rats were infected intratracheally with IRKP to establish a pulmonary infection. (A) Sections of five groups (1: control group, 2: Infected with Kl, 3: Kl þ drug, 4:Kl þ nano and 5:Kl þ combination), lungs obtained after 3, 7 and 14 days and were stained with H&E. Representative images from different areas are shown at 20 power. Note the severe progression of acute lung injury characterized by diffuse alveolar damage in the second group, while stages of improvement after several treatments. B): Sections of five groups of lungs after 14 days and were stained with H&E. Representative images from different areas are shown at 40 power. Note the predominance of alternatively activated macrophages (black arrows) in the second group. While presence of both classically activated macrophages (white arrows) and alternatively activated macrophages in the third and fourth group.
spectroscopy was used to record the formation of AgNPs by reduction of AgNO3 by fungi. The results show strong surface Plasmon resonance centred at 420 nm which indicates the formation of silver nanoparticles, control without silver ions showed no change in colour when incubated under the same conditions. The shape and size of the result particles were elucidated with the TEM. Nanoparticles observed are spherical with a small percentage of elongated particles. It is a variation in particle size, and the average sizes were 27.45, 19.90, 20.84, 15.79, 17.16, 22.62, 18.56 and 12.62 nm for Aspergillus oryzae (RCMB002015), Cladosporium herbarum (RCMB027002), Emericella nidulans (RCMB004002), Fusarium equiseti (RCMB008004), Fusarium proliferatum (RCMB008006), Penicillium echinulatum (RCMB0010042), Trichoderma longibrachiatum (RCMB017007) and Verticillium albo-atrum (RCMB039001) respectively. The obtained mycosynthesized
nanoparticles are in the range of size approximately 1e50 nm and few particles are agglomerated as indicated by Narasimha, JanardhanAlzohairy, Khadri, and Mallikarjuna (2013). Mycosynthesized AgNPs using Verticillium albo-atrum (RCMB039001) as mediator were noticed to have the highest optical density at 420 nm among all tested mycosynthesized AgNPs which indicated the highest concentration of AgNPs beside the least size with range 8.09e16.91 nm and those two indicators are very important for bioactivity of AgNPs, so mycosynthesized AgNPs using Verticillium albo-atrum (RCMB039001) is going to be used for further study. The MIC for imipenem was decreased for IRKP strain. These results demonstrate that Ag-nanoparticles were synergistic with imipenem. This finding was consistent with previous reports that a special synergy effect can occur when antibiotics are combined
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
7
Fig. 5. TEM micrographs of (1) normal lung macrophage after 14 days with normal nucleus and normal organelles, (2) lung macrophage after 14 days of bacterial infection with irregular nucleus and predominance of bacterial cells (white arrow), (3) lung macrophage after treatment with imipenem for 14 days with improved nucleus and presence of bacterial cells (white arrow), (4) lung macrophage after treatment with Ag-nanoparticles (black arrow) for 14 days with improved nucleus and presence of bacterial cells inside cells (white arrow) and (5) cured lung cell after treated with combination Ag-nanoparticles and imipenem (after 14 days).
with an agent that antagonizes bacterial resistance mechanisms (Wagner & Ulrich-Merzenich, 2009). AgNPs themselves at concentrations below 3 mg/L do not display a cytotoxic effect to human cells or blood as well as to environmentally important organisms as was proved in earlier studies (Panacek et al., 2009, 2011; Kvitek et al., 2009;; Krajewski et al., 2013). Our studies demonstrate that AgNPs can interfere with host responses to pulmonary IRKP infection. Combination of AgNPs and imipenem against IRKP pulmonary challenge results in microbial clearance. To assess the activation state of lung cells, we examined effect of imipenem, AgNPs and their combination in tissue of lung after bacterial infection at several time points. Our results suggest that different treatments induce (M1) classically activated macrophages against alternatively activated macrophages (M2). Leopold and Wormley (2014) demonstrated the critical role classically and alternatively activated macrophages during respiratory fungal infections and how the outcome of infection can be determined by the macrophages ability to polarize in a way that supports resolution and not exacerbation of disease. In summary, this study has demonstrated the effects of AgNPs in combination of imipenem to extended to it antibacterial effects against IRKP infection suggesting that future therapeutic strategies with feasible way to improve management of Klebsiella pneumonia infections. References Ahmad, A., Mukherjee, P., Senapati, S., Mandal, D., Khan, M. I., Kumar, R., et al. (2003). Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloids and Surfaces B: Biointerfaces, 28(4), 313e318. s, S., Bened, V. J., & MartArdanuy, C., Lienares, J., Domnguez, M. A., Hernndez-Alle nez-Martnez, L. (1998). Outer membrane profiles of clonally related Klebsiella pneumoniae isolates from clinical samples and activities of cephalosporins and carbapenems. Antimicrobial Agents and Chemotherapy, 42, 1636e1640. Armstrong, G. L., Conn, L. A., & Pinner, R. W. (1999). Trends in infectious disease mortality in the United States during the 20th century. JAMA, 281, 61e66. Balaji, D. S., Basavaraja, S., Deshpande, R., Mahesh, D. B., Prabhakar, B. K., & Venkataraman, A. (2009). Extracellular biosynthesis of functionalized silver nanoparticles by strains of Cladosporium cladosporioides fungus. Colloids and Surfaces B: Biointerfaces, 68(1), 88e92. Bancroft, J. D., & Gamble, M. (2008). Theory and practice of histological techniques (6th ed.). Philadelphia, PA: Churchill Livingstone. Bancroft, J. D., & Stevens, A. L. (1996). Theory and practice of histological techniques (4th ed.). Edinburgh: Churchill Living stone. Basavaraja, S., Balaji, S. D., Lagashetty, A., Rajasab, A. H., & Venkataraman, A. (2008).
Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium semitectum. Materials Research Bulletin, 43(5), 1164e1170. Bhainsa, K. C., & D'Souza, S. F. (2006). Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigatus. Colloids and Surfaces B: Biointerfaces, 47(2), 160e164. Birla, S. S., Tiwari, V. V., Gade, A. K., Ingle, A. P., Yadav, A. P., & Rai, M. K. (2009). Fabrication of silver nanoparticles by phoma glomerata and its combined effect against Escherichia coli, pseudomonas aeruginosa and staphylococcus aureus. Letters in Applied Microbiology, 48, 173e179. Bornet, C., Davin-Regli, A., Bosi, C., Pages, J. M., & Bollet, C. (2000). Imipenem resistance of Enterobacter aerogenes mediated by outer membrane permeability. Journal of Clinical Microbiology, 38, 1048e1052. Boucher, H. W., Talbot, G. H., Bradley, J. S., Edwards, J. E., Gilbert, D., & Rice, L. B. (2009). Bad bugs, no drugs: no ESKAPE! An update from the infectious diseases society of America. Clinical Infectious Diseases, 48, 1e12. Cao, V. T., Arlet, G., Ericsson, B. M., Tammelin, A., Courvalin, P., & Lambert, T. (2000). Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration. Journal of Antimicrobial Chemotherapy, 46, 895e900. nech-Snchez, A., Hernndez-Alle s, S., Martnez-Martnez, L., Bened, V. J., & Dome Albert, S. (1999). Identification and characterization of a new porin gene of Klebsiella pneumoniae: Its role in beta-lactam antibiotic resistance. Journal of Bacteriology, 181, 2726e2732. Fayaz, A. M., Balaji, K., Girilal, M., Yadav, R., Kalaichelvan, P. T., & Venketesan, R. (2010). Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: A study against gram-positive and gram-negative bacteria. Journal of Nanomedicine & Nanotechnology, 6, 103e109. n, N., & Rai, M. K. (2008). Gade, A. K., Bonde, P., Ingle, A. P., Marcato, P. D., Dura Exploitation of Aspergillus niger for synthesis of silver nanoparticles. Journal of Biobased Materials and Bioenergy, 2(3), 243e247. Ghosh, S., Patil, S., Ahire, M., Kitture, R., Kale, S., Pardesi, K., et al. (2012). Synthesis of silver nanoparticles using Dioscorea bulbifera tuber extract and evaluation of its synergistic potential in combination with antimicrobial agents. International Journal of Nanomedicine, 7, 483e496. Gülmez, D., Woodford, N., Palepou, M. F., Mushtaq, S., Metan, G., Yakupogullari, Y., et al. (2008). Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates from Turkey with OXA-48-like carbapenemases and outer membrane protein loss. International Journal of Antimicrobial Agents, 31, 523e526. Hemath, N. K. S., Gaurav, K., Karthik, L., & Rao, K. V. (2010). Extracellular biosynthesis of silver nanoparticles using the filamentous fungus Penicillium sp. Archives of Applied Science Research, 2, 161e167. Hesna, Y., Anderson, G. J., Biddle, J. W., Steward, C. D., Rasheed, J. K., Valera, L. L., et al. (2002). Carbapenem resistance in a clinical isolate of Enterobacter aerogenes is associated with decreased expression of OmpF and OmpC porin analogs. Antimicrobial Agents and Chemotherapy, 46, 3817e3822. Hulkoti, N. I., & Taranath, T. C. (2014). Biosynthesis of nanoparticles using microbes e a review. Colloids and Surfaces B: Biointerfaces, 121, 474e483. Iravani, S. (2014). Bacteria in nanoparticle synthesis: Current status and future prospects. International Scholarly Research Notices, 1e18. Kathiresan, K., Manivannan, M. A., & Nabeel, B. (2009). Studies on silver nanoparticles synthesized by a marine fungus, Penicillium fellutanum isolated from coastal mangrove sediment. Colloids and Surfaces B: Biointerfaces, 71, 133e137. Krajewski, S., Prucek, R., Panacek, A., Avci-Adali, M., Nolte, A., Straub, A., et al. (2013). Hemocompatibility evaluation of different silver nanoparticle concentrations
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002
8
M.M. Abdel-Aziz et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e8
employing a modified chandler-loop in vitro assay on human blood. Acta Biomaterialia, 9, 7460e7468. Kvitek, L., Vanickova, M., Panacek, A., Soukupova, J., Dittrich, M., Valentova, E., et al. (2009). Initial study on the toxicity of silver nanoparticles (nps) against paramecium caudatum. Journal of Physical Chemistry C, 113, 4296e4300. Leopold, W. C. M., & Wormley, F. L. (2014). Classical versus alternative macrophage activation: The ying and the Yang in host defense against pulmonary fungal infections. Mucosal Immunology, 7, 1023e1035. Leying, H., Cullmann, W., & Dick, W. (1991). Carbapenem resistance in Enterobacter aerogenes is due to lipopolysaccharide alterations. Chemotherapy, 37, 106e113. Lin, Y. T., Jeng, Y. Y., Chen, T. L., & Fung, C. P. (2010). Bacteremic community-acquired pneumonia due to Klebsiella pneumoniae: Clinical and microbiological characteristics in Taiwan. BMC Infectious Diseases, 10, 307. Medzhitov, R. (2008). Origin and physiological roles of inflammation. Nature, 454, 428e835. Mizgerd, J. P. (2006). Lung infection public health priority. PLoS Medicine, 3, 76. Moon, J. W., Rawn, C. J., Rondinone, A. J., Love, L. J., Roh, Y., Everett, S. M., et al. (2010). Large-scale production of magnetic nanoparticles using bacterial fermentation. Journal of Industrial Microbiology and Biotechnology, 37, 1023e1031. Muhsin, T. M., & Hachim, A. K. (2014). Mycosynthesis and characterization of silver nanoparticles and their activity against some human pathogenic bacteria. World Journal of Microbiology and Biotechnology, 30, 2081e2090. Naqvi, S. Z. H., Kiran, U., Ali, M. I., Jamal, A., Hameed, A., Ahmed, S., et al. (2013). Combined efficacy of biologically synthesized silver nanoparticles and different antibiotics against multidrug-resistant bacteria. International Journal of Nanomedicine, 8, 187e195. Narasimha, G., Janardhan Alzohairy, M., Khadri, H., & Mallikarjuna, K. (2013). Extracellular syenthesis, characterization and antibacterial activity of silver nanoparticles by Actinomycetes isolative. International Journal of Nanodimens, 4, 77e83. Normann, P., Cuzon, G., & Naas, T. (2009). The real threat of Klebsiella pneumoniae carbapenemase producing bacteria. Lancet Infectious Diseases, 9, 228e236. Panacek, A., Kolar, M., Vecerova, R., Prucek, R., Soukupova, J., Krystof, V., et al. (2009). Antifungal activity of silver nanoparticles against candida spp. Biomaterials, 30, 6333e6340. Panacek, A., Prucek, R., Safarova, D., Dittrich, M., Richtrova, J., Benickova, K., et al. (2011). Acute and chronic toxicity effects of silver nanoparticles (nps) on drosophila melanogaster. Environmental Science and Technology, 45, 4974e4979. Park, J., An, K., Hwang, Y., Park, J. G., Noh, H. J., Kim, J. Y., et al. (2004). Ultra-largescale syntheses of monodisperse nanocrystals. Nature Materials, 3, 891e895. Ravishankar, R. V., & Jamuna, B. A. (2011). Nanoparticles and their potential
application as antimicrobials. Communicating Current Research Technological Advances, 197e209. Reid, G., Jass, J., Sebulsky, M. T., & McCormick, J. K. (2003). Potential uses of probiotics in clinical practice. Clinical Microbiology Reviews, 16, 658e672. Sangeetha, G., Rajeshwari, S., & Venckatesh, R. (2012). Green synthesized ZnO nanoparticles against bacterial and fungal pathogens. Progress in Natural Science: Materials International, 6, 693e700. Shaligram, N. S., Bule, M., Bhambure, R., Singhal, R. S., Singh, S. K., et al. (2009). Biosynthesis of silver nanoparticles using aqueous extract from the compactin producing fungal strain. Process Biochemistry, 44(8), 939e943. Shatzkes, K., Singleton, E., Tang, C., Zuena, M., Shukla, S., Gupta, S., et al. (2016). Predatory bacteria attenuate Klebsiella pneumoniae burden in rat lungs. mBio, 7(6). e01847e16. Soares, A. C., Souza, D. G., Pinho, V., Vieira, A. T., Nicoli, J. R., & Cunha, F. Q. (2006). Dual function of the long pentraxin PTX3 in resistance against pulmonary infection with Klebsiella pneumoniae in transgenic mice. Microbes and Infection, 8, 1321e1329. Soheyla, H., Barabadi, H., Gharaei-Fathabad, E., & Naghibi, F. (2013). Green syntheesis of silver nanoparticles induced by the fungus Penicillium citrinum. Tropical Journal of Pharmaceutical Research, 12, 7e11. Sun, S., Zeng, H., Robinson, D. B., Raoux, S., Rice, P. M., Wang, S. X., et al. (2004). MonodisperseMFe2O4 (M ¼ Fe, Co, Mn) nanoparticles. Journal of the American Chemical Society, 126, 273e279. Vahabi, K., Mansoori, G. A., & Karimi, S. (2011). Biosynthesis of silver nanoparticles by fungus Trichoderma reesei (A route for large-scale production of AgNPs). Insciences Journal, 1(1), 65e79. Verma, V. C., Kharwar, R. N., & Gange, A. C. (2010). Biosynthesis of antimicrobial silver nanoparticles by the endophytic fungus Aspergillus clavatus. Nanomedicine (Lond)., 5(1), 33e40. Wagner, H., & Ulrich-Merzenich, G. (2009). Synergy research: Approaching a new generation of phytopharmaceuticals. Phytomedicine, 16, 97e110. WHO. (2014). Antimicrobial resistance: Global report on surveillance. Yang, D., Guo, Y., & Zhang, Z. (2009). Combined porin loss and extended spectrumlactamase production is associated with an increasing imipenem minimal inhibitory concentration in clinical Klebsiella pneumoniae strains. Current Microbiology, 58, 366e370. Yen, S. C., & Mashitah, M. D. (2012). Characterization of Ag nanoparticles produced by white erot fungi and its in vitro antimicrobial activities. The International Arabic Journal of Antimicrobial Agents, 2, 1e8. Zhang, P., Summer, W. R., Bagby, G. J., & Nelson, S. (2000). Innate immunity and pulmonary host defence. Immunological Reviews, 173, 39e51.
Please cite this article in press as: Abdel-Aziz, M. M., et al., Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem, Journal of Radiation Research and Applied Sciences (2017), https://doi.org/10.1016/j.jrras.2017.09.002