Efficacy of Green Synthesised Iron Oxide Nanoparticles against Various Uropathogens: A Cross-sectional Study

1. Postgraduate, Department of Microbiology, SRM Medical College Hospital and Research Centre, Faculty of Medicine and Health Sciences, Chengalpattu, Tamil Nadu, India. 2. Assistant Professor, Department of Microbiology, SRM Medical College Hospital and Research Centre, Faculty of Medicine and Health Sciences, Chengalpattu, Tamil Nadu, India. 3. Postgraduate, Department of Microbiology, SRM Medical College Hospital and Research Centre, Faculty of Medicine and Health Sciences, Chengalpattu, Tamil Nadu, India. 4. Professor and Head, Department of Microbiology, SRM Medical College Hospital and Research Centre, Faculty of Medicine and Health Sciences, Chengalpattu, Tamil Nadu, India. 5. Assistant Professor, Department of Biotechnology, Amrita School of Biotechnology, Amritavishwa Vidyapeetham, Amritapuri, Clappana, Kollam, Kerala, India. 6. Research Scholar, Department of Microbiology, SRM Medical College Hospital and Research Centre, Faculty of Medicine and Health Sciences, Chengalpa

Abstract

Introduction: The shoot up of antimicrobial resistance leading to the Multidrug Resistance (MDR) phenomenon in clinical pathogens has forced us to develop novel technologies to cease this global threat immediately. Iron oxide nanoparticles can be a breakthrough solution to this dilemma due to its magnetic properties and biocompatibility. Non toxic and biocompatible applications of magnetic nanoparticles can be enriched further by special surface coating with organic or inorganic molecules.

Aim: To determine the antibacterial activity of green synthesised iron oxide nanoparticles against various clinical isolates.

Materials and Methods: This was a cross-sectional study conducted from June 2021 to April 2022. This study was conducted at the Department of Microbiology, SRM Medical College Hospital and Research Centre (SRMMCH&RC), Kattankulathur, Chengalpattu, Tamil Nadu, India. Nanoparticles underwent surface modifications and characterisation using X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray (EDX), Ultraviolet (UV) Visible Absorption Spectra, and Fourier-Transform Infrared Spectroscopy (FTIR) followed by charge characterisation through agarose gel electrophoresis. Kirby-Bauer Disc Diffusion method was used for screening the sensitivity and resistance pattern of 50 selected isolates and Minimum Inhibitory Concentration (MIC) was assessed using MIC Microbroth Dilution technique with the help of resazurin. Tukey post-hoc multiple comparisons test to analyse the zone of inhibition of antibacterial efficacy.

Results: Out of the four different concentrations of bare and coated nanoparticles (0.0375 mg/mL, 0.07 mg/mL, 0.15 mg/mL, 0.3 mg/mL), bare nanoparticles inhibited the growth of Methicillin Resistant Staphylococcus aureus (MRSA) at 0.3 mg/mL while citrate coated nanoparticles inhibited the growth at 0.15 mg/mL, 0.018 mg/mL, 0.0375 mg/mL, 0.07 mg/mL, and 0.15 mg/mL dilutions were used in case of Carbapenem-resistant Klebsiella pneumoniae (CR K. pneumoniae) and MDR Escherichia coli, from which both organisms were inhibited at 0.15 mg/mL of bare and coated nanoparticles.

Conclusion: Iron nanoparticles synthesised from the marine algae Chaetomorpha antennina could be used in the future as a drug carrier or as an antimicrobial agent.

Antibacterial activity, Iron oxide nanoparticles, Minimum inhibitory concentration, Trisodium citrate

The inception and expansion of pathogens with new mechanisms of drug resistance are continuing to intimidate our ability to diagnose and treat common infections. Extensive use of antibiotics and misuse of them are the prime reason for antibiotic resistance in bacteria (1). Among which MRSA, MDR E. coli, CR K. pneumoniae are the significant global health issue that causes morbidity and mortality worldwide. In recent years, MRSA has led to many nosocomial infections responsible for deadly conditions like necrotising fasciitis, osteomyelitis, sepsis, toxic shock syndrome and endocarditis. Penicillin-binding protein (PBP2a) encoded by mecA or mecC is accountable for MRSA. Vancomycin has been used as the choice of drug for decades but certain isolates with complete or intermediate resistance have emerged recently (2).

One of the most frequent pathogens encountered in clinical settings is E. coli, which is associated with nosocomial and community acquired infections, now it has developed resistance to the broad spectrum of antimicrobial agents. Many strains of E. coli are producers of β-lactamases such as Extended Spectrum β-Lactamases (ESBL), AmpC β-lactamases and Metallo β-Lactamases (MBL). The Centre for Disease Control and prevention (CDC) defines MDR as the ability of an organism to be resistant to atleast one agent in three or more antimicrobial groups (3). Among these, the most common one is MDR E. coli (4).

Currently, Klebsiella pneumoniae is an important cause of healthcare-associated infections. Many strains carry plasmids that encode MDR due to the synthesis of carbapenemases and MBL. Due to the production of these enzymes, antibiotics coming under the carbapenem group cannot work efficiently. In this condition, the polymixin and colistin is used as the last line of therapy. Moreover, colistin resistant strains are increasing day by day (5).

An important area of modern science is nanotechnology, which is concerned with the study of and manipulation of matter at a scale between 1 and 100 nanometers (6). Synthesis of nanoparticles using biosystems such as plants, algae, bacteria, yeast, fungi and actinomycetes were termed as green synthesis. The perks of green nanotechnology is an alternative, well organised, economical, and eco-friendly method for the synthesis of nanoparticles with specified properties (7). Because of their magnetic properties and biocompatibility, iron oxide nanoparticles have been widely used as bacterial separation agents, contrast agents for bio imaging, and magnetic hyperthermia agents. They can lose their surface properties when exposed to the environment and can result in particle aggregation, change in charge and other surface properties. Therefore, surface modification using sodium citrate will be useful to avoid such problems (8). This study tries to utilise the ability of nanoparticles to disrupt the bacterial cell by the production of Reactive Oxygen Species (ROS) (9). Since, urinary tract infections are the most common nosocomial infections and the selected organisms were commonly associated with such infections, this study focuses on the evaluation of antibacterial activity of iron oxide nanoparticles against uropathogens such as MRSA, CR K. pneumoniae and MDR E. coli.

Material and Methods

This cross-sectional study was conducted in the Department of Microbiology, SRM Medical College Hospital and Research Centre (SRMMCH&RC), Kattankulathur, Chengalpattu, Tamil Nadu, India. A total of 50 resistant isolates were isolated from urine specimens collected from SRMMCH&RC during a total time period of June 2021 to April 2022. Out of which eight MRSA, 22 CR K. pneumoniae and 20 MDR E. coli were obtained. The study was approved by the Institutional Ethical Committee (IEC) and ethical clearance was obtained (IEC.No 2898/IEC/2021). Similar studies were insufficient in order to calculate sample size, so authors collected as much as isolates from hospital (50 resistant isolates from urine samples during the mentioned period). As only bacterial isolates were utilised, individual patients’ consent were not required for the study.

Inclusion criteria: Fifty (50) MRSA, CR K. pneumoniae, and MDR E. coli isolates from urine samples collected before initiation of antibiotic therapy were included in this study.

Exclusion criteria: Isolates other than MRSA, CR K. pneumoniae, and MDR E. coli from clinical samples isolates collected after the initiation of antibiotic therapy were excluded in this study.

Synthesis of Nanoparticles

Synthesis of iron oxide nanoparticles from the marine green algae Chaetomorpha antennina was done by using previous study conducted at Amrita School of Biotechnology, Kollam Kerala, India, 0.1 g of algal powder along with ultrapure water was heated to 70-80°C. The crude algal extract was centrifuged and the supernatant thus obtained was used as the seaweed bioextract. 0.1 M FeCl₂.4H₂O and 0.1% algal extract were added in the ratio of 2:3, in four different sets, each with a different pH. The pH of the reactions was adjusted to 6, 8, 10, and 12 using NaOH and the effect of pH on the nanoparticle properties were studied. These reactions were maintained at temperatures ranging from 60-70°C. The synthesised nanoparticle was washed thrice using 70% ethanol. It was kept in a hot air oven for 24 hours and dried. To evaluate the effect of temperature on the nanoparticle synthesis, two different sets of reactions were performed at a temperature ranging from 60-70°C and at room temperature. The green synthesised nanoparticle was washed and dried for storage (10).

Surface modification of iron oxide nanoparticles: The nanoparticle was coated with trisodium citrate (Na₃C₆H₅O₇) (0.01 M) by adding the trisodium citrate solution to the sonicated bare FeNp. Sonication were performed frequently to disperse nanoparticles into water (10).

Characterisation of Nanoparticles

Characterisation of the Fe₃O₄ nanoparticles was undertaken by the following methods.

UV-visible absorption spectra: Shimadzu’s UV-2540s equipped with an ISR-240A sphere assembly were used to measure UV-visible absorption spectroscopy measurements in aqueous buffer in the range 300-800 nm. A quartz cuvette with a path length of 1 cm was used to scan the absorbance from 190-900 nm.

X-ray diffraction: With the help of X-ray powder diffraction, the phase composition of Fe₃O₄ nanoparticles was investigated over the range of 20-600 at a rate of five nanometers per minute (14, 1.54060 A). Using the Debye-Scherrer equation, one can predict the average particle size of Fe₃O₄ nanoparticles by relating its peak broadening in XRD to particle size. The following equation illustrates this relationship:

d=λk/cosθxβ

Fourier Transform Infrared Spectroscopy (FTIR): The Fe₃O₄- NP characteristics like surface bond were analysed using a FTIR spectrometer.

Scanning Electron Microscopy (SEM): A SEM study was carried out on nanoparticles synthesised at pH 8 and structural data was recorded and it enables the visualisation of nanoparticle’s morphological characteristics.

Characterisation of FeNp based on charge: The charge of Fe₃O₄ -nanoparticles was determined using Agarose Gel Electrophoresis (AGE). 1X Tris-acetate-EDTA (Ethylenediaminetetraacetic Acid) buffer was used to prepare a 2.5 percentage of agarose gel. A 100 V voltage was applied to the gel to run the samples (11).

Selection of isolates using Antibiotic Susceptibility Test (AST): Clinical isolates such as MRSA, MDR E. coli and CR K. pneumoniae are screened using the Kirby-Bauer technique on Mueller-Hinton agar (12). The Clinical and Laboratory Standards Institute (CLSI) guidelines for 2021 were used to interpret the zone sizes (3).

Minimum Inhibitory Concentration (MIC) evaluation: The MIC of green synthesised iron oxide nanoparticles (Fe₃O₄) was evaluated using the broth microdilution technique with the help of 96 microtitre plates along with resazurin dye (0.015%). The 1/150 dilution of bacterial suspension were prepared from 0.5 McFarland standardised inoculum in Mueller-Hinton broth and 50 μL of this solution was transferred to each well except first column of microtitre plate (keep the first column of microtitre plate empty). A 3 mg/mL and 6 mg/mL nanoparticle:distilled H₂O solution was formulated for enterobacteriaceae and MRSA respectively (13).

Statistical Analysis

Tukey post-hoc multiple comparisons test to analyse the zone of inhibition of antibacterial efficacy.

Results

A total of 50 isolate comprising 8 (16%) MRSA, 22 (44%) CR K. pneumoniae, and 20 (40%) MDR E. coli were isolated from the laboratory. Male to female ratio was found to be 6:2, 9:13, and 12:8 for MRSA, CR K. pneumoniae and MDR E. coli respectively. The maximum number of CR K. pneumoniae was isolated from the Medical Intensive Care Unit (MICU) ward 7 (31.8%) followed by Urology 4 (18.2%), General Medicine 4 (18.2%), Coronavirus Disease-2019 (COVID-19) Intensive Care Unit (ICU) 2 (9.1%), Electronic Intensive Care Unit (EICU) 2 (9.1%), Respiratory ICU 1 (4.5%), Labour ward 1 (4.5%) and casualty 1 (4.5%). Most of the MRSA was isolated from the MICU 4 (50%) followed by Surgical Intensive Care Unit (SICU) 3 (37.5%) and Respiratory Intensive Care Unit (RICU) 1 (12.5%). Majority of MDR E. coli were isolated from General Medicine 7 (35%) followed by Nephrology 3 (15%), Casualty 3 (15%), General Surgery 3 (15%), Urology 2 (10%), Neuroscience Intensive Care Unit (NSICU) 1 (5%) and MICU 1 (5%) (Table/Fig 1).

Antibiotic resistance pattern: The MRSA had the highest resistance to penicillin (100%), cefoxitin (100%) bacitracin (100%), and erythromycin (87%) and was susceptible to antibiotics such as novobiocin, clindamycin, tetracycline, linezolid, chloramphenicol, and vancomycin. CR K. pneumoniae showed resistance to most of the antibiotics and had 100% resistance to antibiotics of group penicillins, β lactams+β lactam inhibitors, 1^st generation cephalosporins, carbapenems and aminoglycosides. The lowest resistance was observed in nitrofurans (68%), sulphonamides (68%) and cefuroxime (68%). All the MDR E. coli isolates were AmpC producers and carbapenem-resistant. They showed 100% resistance to β lactams, β lactam+inhibitors, all generation cephalosporins, and carbapenems except meropenem. The lowest resistance was observed in aminoglycosides and nitrofurans (Table/Fig 2),(Table/Fig 3),(Table/Fig 4).

Iron Nanoparticle Characterisation

UV-visible absorption spectra: The Surface Plasmon Resonance (SPR) of bare nanoparticles was found in between 190 and 250 nm which corresponds to that of magnetite. Near-infrared absorption indicates the complex to be Fe₃O₄, since Fe₂O₃ which also has
two ionic species does not absorb in the near-infrared region. The presence of carbon nanoparticles could be appreciated using the presence of a transient absorption band formed at wavelengths between 360 and 320 nm, 320 and 290 nm, 290 and 270 nm and 270 and 250 nm.

Fourier-transform infrared spectroscopy: Waves at 3,319 cm^-1 correspond to OH stretching. The absorption band at 1,641 cm^-1 indicates the presence of the C=O group around the nanoparticles. So, the synthesised nanoparticles were characterised as magnetite, since two absorption bands were observed at 307 cm^-1 and 535 cm^-1.

X-ray diffraction: The angle between the transmitted and reflected beam was observed as 35.25° which is complementary to the crystallographic value of (311) of crystalline Fe₃O₄-nanoparticles. The results observed were highly acceptable and comparable with the standard value and sizes of magnetite nanoparticles. Hence, the crystallite size of the green synthesised iron oxide nanoparticles was identified as 6.625 nm using XRD along with the application of the Scherrer equation (Table/Fig 5).

Scanning electron microscopy: The average size of the nanoparticle was identified as 24 nm (Table/Fig 6),(Table/Fig 7).

Charge characterisation of FeNp: Charge characterisation of FeNp was done using 2.5% AGE and the surface charge of prepared nanoparticles was identified as positive for bare iron oxide nanoparticles and negative for citrate coated (capped) iron oxide nanoparticles.

Minimum inhibitory concentration evaluation: Four different concentrations of bare and capped nanoparticles (0.0375 mg/mL, 0.07 mg/mL, 0.15 mg/mL and 0.3 mg/mL) were used to determine the MIC value against MRSA isolates while 0.018 mg/mL, 0.0375 mg/mL, 0.07 mg/mL, and 0.15 mg/mL dilutions were used in case of CR K. pneumoniae and MDR E. coli.

MIC against Methicillin-resistant Staphylococcus aureus isolates (MRSA): The MIC determination of green synthesised Fe₃O₄ nanoparticles against MRSA isolates. Wells extending from columns 2 to 5 and row A to F and wells extending from columns 7 to 10 and row A to F contains different MRSA isolates with varying concentration of bare and capped nanoparticles respectively. Column 1 and 6 (row A to F) shows nanoparticles control with no organism. Columns 1 to 5 (row G) were positive controls that contain suspensions of bacterial isolates alone. Row H (column 1 to 5) and row H (column 10 to 12) were resazurin and citrate control respectively with no contamination. Since, colour change occurred in column 2 in the case of bare nanoparticles and column 8 in the case of capped nanoparticles, the MIC value is confirmed as 0.3 mg/mL for bare nanoparticles and 0.15 mg/mL for capped nanoparticles (Table/Fig 8).

MIC against MDR E. coli and CR K. pneumoniae isolates: Wells extending from columns 2 to 5 and row A to F and wells extending from columns 7 to 10 and row A to F contains isolates with varying concentration of bare and capped nanoparticles respectively. Column 1 and 6 (row A to F) shows nanoparticles control with no organism. Row G (columns 1 to 5) were positive controls that contains suspensions of bacterial isolates alone. Row H (column 1 to 5) and row H (column 10 to 12) were resazurin and citrate control respectively with no contamination. Since, colour change occurred in column 2 in the case of bare nanoparticles and column 7 in the case of capped nanoparticles, the MIC value is confirmed as 0.15 mg/mL for bare and capped nanoparticles in the case of both isolates (Table/Fig 9),(Table/Fig 10).

Discussion

Antibiotic resistance has become a hurdle that intimidates our ability to treat common infections. Heavy dose and combination therapies are the current treatment option against these pathogens. The rise in resistant pathogens against these combination drug therapies in the past decade has made them ineffective. A high dosage of the antibiotic does not turn out to be helpful in case of patients with co-morbidities and low immunity as seen in older individuals, so it is important to introduce novel techniques like green nanotechnology to exploit the possibilities of overcoming this crisis.

Out of 50 urine samples, CR K. pneumoniae (22), MDR E. coli (20), and MRSA (8) were isolated. Since, MRSA pathogens are commonly associated with hospital-acquired infections, the number of isolates was comparatively less and most of them are from ICUs. Similar trends were reported by Sanjana RK et al., and Mallick SK and Basak S, in their studies conducted in Nepal and central India (14),(15).

The maximum number of CR K. pneumoniae and MRSA was isolated from the age group above 60, were 63.5% and 62.5% respectively. However, in the case of MDR E. coli, the maximum number of isolates were from the age group 19 to 60 (65%). Lohan K et al., and Chellapandi K et al., found similar results in their studies (16),(17).

In the case of MDR E. coli and CR K. pneumoniae, maximum isolates were from the General Medicine (GM) ward (30%). Kapoor G and Chaurasia D, in their study, revealed the same results of CR K. pneumoniae but it does not coincide with the results of MDR E. coli (18). Since, the number of patients admitted in GM was considerably high, the chance of the spread of bacterial infection rises. The reasons behind CR K. pneumoniae and MRSA infection in ICU patients might be a result of prolonged hospitalisation, catheterisation, lack of surveillance and bundle approach.

While taking the antibiogram of MRSA into consideration, all eight isolates showed (100%) resistance to antibiotics such as penicillin, cefoxitin and bacitracin. A 50% of isolates were resistant to ciprofloxacin and 87% were resistant to erythromycin. All MRSA isolates were found sensitive to tetracycline, linezolid, chloramphenicol and nitrofurantoin. Variations in antibiogram patterns were reported in certain studies conducted by Lohan K et al., (16). These variations might be due to the demographical changes pertaining to the geographical area and strains used.

However, the increasing resistance of E. coli and CR K. pneumoniae were in consideration CR K. pneumoniae showed resistance to most of the antibiotics and had 100% resistance to antibiotics of group penicillins, β lactams+β lactam inhibitors, 1st generation cephalosporins, carbapenems and aminoglycosides. Lowest resistance was observed against nitrofurans (68%), Sulphonamides (68%) and cefuroxime (68%). These results can be positively correlated with previous studies conducted by Indrajith S et al., (19). Dissimilarity in resistance percentage might be a result of the change in the number of isolates.

Most of the MDR E. coli isolates were AmpC producers and carbapenem-resistant. They showed 100% resistance to penicillins, β lactam+β lactam inhibitors, 1^st 2^nd 3^rd, and 4^th generation cephalosporins, carbapenems except for meropenem (95%) and fluoroquinolones {(Ofloxacin (95%)}. Lowest resistance was observed against aminoglycosides {(Amikacin (45%) and gentamicin (60%)}, Tetracycline (55%) and nitrofurantoin (0%). The higher resistance to these antibiotics was reported in other study conducted by Nosheen S et al., in paediatric patients (20). Contradictory results in few antibiotics were observed in a study conducted by Kibret M and Abera B (21). Moreover, the difference in strains can be a reason for these results.

This study tries to utilise the ability of nanoparticles to disrupt the bacterial cell by the production of Reactive Oxygen Species (ROS), for considering the implementation of nanotechnology in antimicrobial therapy. The green synthesised Iron Oxide Nanoparticles (IONPs) and the citrate coated IONPs were characterised by FTIR spectrometry, XRD, SEM and UV-visible absorption spectra. As a result of XRD, 35.25° diffraction peaks are evident that accord with the crystal plane of (311) of crystalline Fe₃O₄ nanoparticles. The crystallite size of the magnetic Fe₃O₄ was found to be 6.625 nm. Studies conducted by Mahdavi N et al., corroborate these results (11). In present study, during UV-visible absorption spectra the characteristic surface plasmon band is centered at 190-250 nm and the presence of nearby infrared absorption confirmed that the complex is Fe₃O₄. The presence of transient absorption bands between the wavelengths 360-320, 320-290, 290-270, and 270-250 nm also substantiated the formation of carbon functionalised nanoparticles. Four infrared bands (3319 cm^-1, 1641 cm^-1, 535 cm^-1, and 307 cm^-1) observed in FTIR in which signal at 3319 cm^-1 corresponds to OH stretching. The charge of bare and surface modified nanoparticles was identified using AGE as positive and negative respectively. In 2016 Krohling CA et al., got similar results in their study of iron homeostasis (22).

Different concentrations of both bare and capped Fe3O4 nanoparticles were used to determine MIC against MRSA, MDR E. coli, and CR K. pneumoniae. MIC micro broth dilution using resazurin dye was used for this purpose. Out of the four dilutions, bare nanoparticles inhibited the growth of MRSA at 0.3 mg/mL while citrate coated nanoparticles inhibited the growth at 0.15 mg/mL. The reason behind this observation might be due to the better absorption of citrate coated nanoparticles as the MRSA are citrate utilising organisms. Production of siderophores by MRSA gives an added aid in Fe₃O₄ nanoparticles absorption (23). Validating results were appreciated in other studies done by Cavassin ED et al., in the year 2015 (24).

Four different concentrations of bare and capped nanoparticles were tested with MDR E. coli and CR K. pneumoniae from which both organisms were inhibited at 0.15 mg/mL of bare and capped nanoparticles. The varying results while compared with MRSA might be because of the non utilisation of citrate by MDR E. coli and the presence of capsular proteins in K. pneumoniae (25),(26). Hence, this study revealed that green synthesised iron oxide nanoparticles have antibacterial activity against organisms such as MRSA, MDR E. coli, and CR K. pneumoniae isolated from urine samples of patients diagnosed with urinary tract infections. Considering that very few in-vitro and in-vivo studies have been conducted on the antimicrobial activity of citrate coated iron oxide nanoparticles, further research is needed to exploit nanotechnology to overcome antimicrobial resistance.

Limitation(s)

In-vitro studies have the limitation of laboratory and clinical setup errors. Further investigation on molecular mechanisms governing the antimicrobial activity of nanoparticles was not included in this study.

Conclusion

Overuse and misuse of antibiotics have led to the spread of antimicrobial resistance globally and are on an unpredictable rise. The need for a novel lead molecule for the discovery and development of a new drug class is a high priority action to be undertaken in the field of microbiology and pharmacology. Adopting nanoparticle science as an aid to overcome this global hazard could bear fruitful results in the future. This study has successfully tapped into the possibility of utilising nanotechnology in the near future as perceptible susceptibility was detected for these pathogens towards iron oxide nanoparticles. Fe₃O₄ nanoparticles can act as a remarkable carrier for antibiotics and could be considered a serious contender in the development of new antibiotic molecules.

Acknowledgement

Sincere, thanks to Dr. KV Leela from SRM Medical College Hospital and Research Centre for their valuable guidance. Author thank Dr. Dalia Vishnudasan, Mr. Sujith R, Mr. Balamurali, Mr. Jayaprakash and Ms. Shubhangi Trisal, for their kind support throughout the project work. Authors acknowledge Sir C.V Raman Research Park and Amrita School of Biotechnology for the provision of resources and research amenities.