INTRODUCTION

The daily increase in life-threatening infections caused by multi-resistant microorganisms (especially bacteria) leading to an increase in deaths globally has inspired many researchers to search for antimicrobial agents to develop new antibiotics from medicinal plants (Basappa et al., 2013; Betts, Hornsey, & Ragione, 2018). Medicinal plants contain phytochemicals in a crude form that, when biosynthesized into nanoparticles, may exert better therapeutic effects. Medicinal plants' eco-friendliness and cost-effectiveness have increased their popularity for the biological synthesis of silver nanoparticles and nanomaterials (Aritonang, Koleangan, & Wuntu, 2019). Plant-mediated nanoparticle synthesis is getting more popular because of the high reactivity of plant extract and easy availability of plant materials. In a comparative in-vitro and in-vivo toxicity study (Kalaichelvan, Manjumeena, Girilal, & Mahesh, 2013), it has been concluded that biologically conjugated AgNPs are less toxic than chemically conjugated AgNPs. This method of nanoparticles synthesis involves no toxic chemicals and has been termed as green chemistry procedure as opined by (Kalaichelvan et al., 2013).

Studies on AgNPs and other nanomaterials have increased in the post-antibiotic era to search for new agents that can help combat pathogenic microorganisms without promoting the appearance of new resistances (Betts et al., 2018). The exceptional antibacterial activity exhibited by AgNPs has drawn the attention of researchers and industries on this nanomaterial. AgNPs have shown antimicrobial activity against various infectious and pathogenic microorganisms, including multidrug-resistant bacteria (Siddiqi, Husen, & Rao, 2018). The potential and effectiveness of AgNPs as antibiotics alternative and their efficiency even against multidrug-resistant organisms is related to their various mechanisms of action, which attack microorganisms in multiple structures at a time and give them the ability to kill various types of bacteria (Cheng et al., 2016; Lee, Ko, & Hsueh, 2019). The infections caused by antibiotic-resistant microorganisms have profound public health implications and are a global concern. Therefore, the emergence of AgNPs as an excellent alternative has become beneficial as they can be applied to prevent infections caused by these microorganisms, decontaminate medical supplies and even combat infections in the course (Betts et al., 2018; Natan & Banin, 2017). This study focuses on the characterization and rate of killing conjugated silver nanoparticles against selected clinical bacterial isolates.

MATERIALS AND METHODS

Collection, Identification and Confirmation of Clinical Bacterial Isolates

The Escherichia coli, Pseudomonas fluorescens, Klebsiella pneumoniae, Salmonella typhimurium used were obtained from the Microbiology Department of General Hospital, Minna. They were Gram-stained, and their biochemical tests were carried out in order to identify them according to the methods of (Cheesbrough, 2010) and then subcultured into Nutrient Agar slants for molecular characterization for confirmation (Incorporation, 2012; Incorporation, 2016; NCBI, 1988) and kept in a refrigerator until they were ready for further use.

Biosynthesis of Silver Nanoparticles from Aqueous Crude Extract of Whole Plant of E. heterophylla

The biosynthesis of silver nanoparticles from the aqueous crude extract of the whole plant of E. heterophylla was carried as outlined in the modified method of (Kasthuri, Kathiravan, & Rajendiran, 2009). The stock solution of 80 mg/ml was prepared from the aqueous crude extract of the whole plant of E. heterophylla. A 0.5 ml aliquot was measured from the stock solution and added to 2.5 ml of 1 mM AgNO3 solution in a test tube, then buffered to the various pH levels of 4, 5, 6, 7, 8, 9, 10 and 11. It was stabilized using 600 molecular weight of poly ethylene glycol (PEG 600) and then made up to the final volume of 5 ml with NaOH and HCl solutions buffers. The test tubes were covered with aluminium foil to avoid photoreduction of silver ions and then incubated at 37oC under agitation (200 rpm) and dark condition for 36 h. In silver nanoparticle formation, the solution turned from yellowish to bright yellow and dark brown.

Characterization of Conjugated Silver Nanoparticles   

Ultraviolet-Visible Spectroscopy of Conjugated Silver Nanoparticles

The ultraviolet-visible spectroscopy of the CAgNPs was determined from 300-700 nm using a UV-Visible Spectrophotometer 20D, Techmel and Techmel, USA (Kasthuri et al., 2009).

Size Distribution of Conjugated Silver Nanoparticles

The size distribution of the CAgNPs was determined using Malvern Zetasizer version 7.01.

Fourier Transform Infra-Red (FTIR) Spectroscopy

The analysis of the functional group of the bio-reducing agent present in the extract (80 mg/ml) was measured by FTIR. A small aliquot of the concentrated reaction mixture was measured in the transmittance mode at 500 to 4500 cm-1 after the reaction. The extract spectra were taken after the biosynthesis of silver nanoparticles was determined (Sermakkani & Thangapandian, 2012).

Energy dispersive spectroscopy (EDS)

The energy dispersive X-ray spectrometry carried out an elemental compositional analysis on the sample (EDS) attached with Scanning Electron Microscope (SEM) Machine. THE SEM MACHINE SERVED the EDS analysis of the Ag sample (Sermakkani et al., 2012).

Transmission electron microscope (TEM)

To determine the shape and surface morphology, TEM was utilised. Thin films of the sample (conjugated AgNPs solution) were prepared by placing a minimal amount on the carbon-coated grids. The films on the TEM grids were allowed to stand for 2 minutes, and the extra solution was removed using blotting paper, and the grid was then allowed to dry overnight before the measurement was taken. The TEM measurements were carried out on a JEOL model 1200EX instrument, operated at an accelerating voltage at 120 kV (Sermakkani et al., 2012).

Microbiological activity

Standardization of Clinical Bacterial Isolates

The clinical isolates' population was determined from the 0.5 McFarland Turbidity Standard prepared (Mcfarland, 1907; Murray, Baron, Jorgensen, Landry, & Pfaller, 2007).

Determination of the Rate of Killing

The rate of killing of the CAgNPs and control (without CAgNPs) against the clinical bacterial isolates was determined by the modified method of (Hugo & Russell, 2000; Oloninefa, Abalaka, & Daniyan, 2020). A standardized overnight culture with a 1.5 × 108 cfu/ml population was used. A 0.20 ml of the inoculum was added to 10 ml of the conjugated AgNPs. A 1.0 ml of the mixture was withdrawn at intervals of 1, 2, 3, 4, 5, 6 and 7 h respectively and diluted tenfold (10-1), then plated on Mueller Hilton Agar in duplicates incubated at 37oC for 24 h. The rate of killing the clinical bacterial isolates by the control was determined by adding 0.20 ml of the inoculum to 10 ml of Nutrient Broth. A 1.0 ml of the mixture was withdrawn at intervals of 1, 2, 3, 4, 5, 6 and 7 h respectively and diluted tenfold (10-1), then plated on Mueller Hilton Agar in duplicates incubated at 37oC for 24 h. The population of the clinical isolates was counted and expressed in log10 CFU/ml after the exposure to the CAgNPs and control, respectively.

Data Analysis

The data obtained from the study were subjected to analysis of variance (ANOVA) using IBM SPSS Statistics Version 23 and Microsoft Excel 2010. All data were expressed as mean ± standard error of the mean. The values with different superscripts along the same column were significantly different (P < 0.05).

RESULTS

Biochemical characteristics of the clinical isolates

The result of the Gram stain reactions and biochemical tests conducted on the four clinical bacterial isolates is shown in Table 1. Triple sugar iron (TSI), methyl red, indole, catalase citrate utilisation and urease were carried out on the isolates for identification. The organisms identified and later confirmed by molecular characterization were: E. coli strain MRE 600 (CP014197.1), Salmonella enteric subsp. enteric serovar typhi PMO 16/13 (CP12091.1), K. pneumoniae strain HZW25 (CP025211.1) and P. fluorescens strain 2P24 (CP025542.1) (Table 2).

Table 1

Gram reactions and biochemical characteristics of the clinical bacterial isolates

Tests

A

B

C

D

Gram Reactions

-

-

-

-

TSI

A/A, G (+), H2S (-)

K/A, G (+), H2S (+)

A/A, G (+), H2S (-)

K/K, G (-), H2S (-)

Methyl Red

+

-

+

-

Indole

+

-

-

-

Catalase

+

+

+

+

Citrate Utilisation

-

+

-

+

Urease

+

+

-

+

Identity

E. coli

S. typhimurium

K. pneumoniae

P. fluorescens

[i] + = Positive; - = Negative; TSI= Triple Sugar Iron; K= Alkaline; A= Acid; G= Gas; H2S= Hydrogen sulphide

Table 2

Molecular Characterization of Clinical BacterialIsolates

Clinical Isolates

Max Score

Total Score

Query Cover

Expected Value

Identity

Accession Number

E. coli strain MRE 600

2660

16064

100%

0.0

100%

CP014197.1

P. fluorescens strain 2P24

2689

2689

100%

0.0

100%

CP025542.1

Salmonella enteric subsp. enteric serovar typhi PMO 16/13

3546

3546

100%

0.0

100%

CP12091.1

K. pneumoniae strain HZW25

797

6348

100%

0.0

98%

CP025211.1

UV/Visible Spectrophotometry of Conjugated Silver Nanoparticles

The result of UV/Visible spectrophotometry of conjugated silver nanoparticles determined between pH 4-11 is shown in Table 3. The maximum wavelength obtained was 410.33 nm, while the absorbance value ranges from 0.5333-0.986.

Table 3

UV/Visible Spectrophotometry of Conjugated Silver Nanoparticles

pH

Wavelength

Absorbance

4

410.33±0.333a

0.533±0.003a

5

410.33±0.333a

0.774±0.003c

6

410.33±0.333a

0.821±0.005d

7

410.33±0.333a

0.711±0.005b

8

410.33±0.333a

0.923±0.012e

9

410.33±0.333a

0.986±0.003f

10

410.00±0.333a

0.975±0.003f

11

410.33±0.333a

0.779±0.005c

[i] Results represent mean ± standard error of the mean of triplicate determination. The values with the same superscript in the same column are not significantly different at p<0.05

Size Distribution of Conjugated Silver Nanoparticles

Figure 1 shows the size distribution of the CAgNPs. The CAgNPs had a Zeta-average of 237.8 d.nm.

Figure 1

Size Distribution of Conjugated AgNPs.

Conjugated AgNPs- Aqueous extract of whole plant of Euphorbia heterophylla + 1mM Silver nitrate solution + PEG 600 (Zeta average = 237.8 d.nm)

https://www.nrfhh.com/f/fulltexts/145339/image1_min.jpg

Fourier Transform-Infra Red of Conjugated Silver Nanoparticles

The results of FTIR spectra in Figure 2 reveal the biomolecules responsible for the stability and reduction of silver ions exposed to the aqueous extract of the whole plant of Euphorbia heterophylla. The FTIR spectra reveal the peaks at 1635.66 cm-1 and 3308.94 cm-1 which correspond to alkenes (C=C) and alcohol (O-H).

Figure 2

Transform-Infrared Spectroscopy of CAgNPs

https://www.nrfhh.com/f/fulltexts/145339/image2_min.jpg

Transmission Electron Microscopy of Conjugated Silver Nanoparticles

Figure 3 show the morphology of the CAgNPs at 50 nm. The CAgNPs were monodispersed and spherical.

Figure 3

Transmission Electron Microscopy of CAgNPs

https://www.nrfhh.com/f/fulltexts/145339/image3_min.jpg

Energy Dispersive Microscopy (EDS) of CAgNPs

Figure 4 shows that CAgNPs contained silver and had 3.0 keV of silver when the EDS was carried out. The presence of carbon and copper in the spectra was part of the materials used during the sample preparation.

Figure 4

4: Energy Dispersive Spectroscopy of Conjugated AgNPs

https://www.nrfhh.com/f/fulltexts/145339/image4_min.jpg

Rate of Killing

Figure 5 shows the results of the rate of killing of CAgNPs and control when clinical bacterial isolates: E. coli strain MRE 600 (CP014197.1), Salmonella enteric subsp. Enteric serovar typhi PMO 16/13 (CP12091.1), K. pneumoniae strain HZW25 (CP025211.1) and P. fluorescens strain 2P24 (CP025542.1) were exposed for 7 h. There was a reduction in the E. coli strain MRE 600 (CP014197.1) population from 2.00-1.31log10cfu/ml when exposed to CAgNPs between 1-5 h while 0.00log10cfu/ml was obtained between 6-7 h. There was an increase in the E. coli strain MRE 600 (CP014197.1) population from 11.39-14.64log10cfu/ml when exposed to the control for 1-7 h. On the other hand, a reduction in populations of Salmonella enteric subsp. enteric serovar typhi PMO 16/13 (CP12091.1) from 2.18-1.49log10cfu/ml was recorded between 1-6 h but 0.00log10cfu/ml was obtained for 7 h when exposed to CAgNPs. However, an increment in the populations of Salmonella enteric subsp. Enteric serovar typhi PMO 16/13 (CP12091.1) from 10.96-12.46log10cfu/ml was obtained when exposed to the control for 1-7 h (Figure 5).

Furthermore, there was a reduction in the populations of K. pneumoniae strain HZW25 (CP025211.1) from 11.67-11.10log10cfu/ml when exposed to CAgNPs between 1-7 h, while there was an increase in the populations of K. pneumoniae strain HZW25 (CP025211.1) from 11.72-11.98log10cfu/ml when exposed to the control for 1-7 h. In addition, there was a reduction in the populations of P. fluorescens strain 2P24 (CP025542.1) from 3.13-2.52log10cfu/ml when exposed to CAgNPs between 1-7 h. On the other hand, increment in the populations of P. fluorescens strain 2P24 (CP025542.1) from 10.59-11.56log10cfu/ml was recorded when exposed to the control for 1-7 h (Figure 5).

Figure 5

Rate of Killing of Conjugated Silver Nanoparticles and Control

https://www.nrfhh.com/f/fulltexts/145339/image5_min.jpg

DISCUSSION

The maximum wavelength obtained (410.33 nm) when ultraviolet-visible spectroscopy of conjugated silver nanoparticles was determined in line with the studies carried out by Kasthuri et al. (2009); Malawong et al. (2021), which obtained similar results (Table 2). The size distribution of CAgNPs revealed an average size of 237.8 d.nm and was confirmed by the TEM image shown in Figure 3. As opined by (Xueting et al., 2018), the size of nanoparticles pave the way for easy penetration of silver ions into the bacterial cell and react with DNA molecule leading to the collapse of DNA replication which eventually causes bacterial cell dysfunction. The result of the FTIR spectrum showing the presence of peak 3308.94 cm-1 (O-H) may be responsible for stabilising and reducing the silver ions when exposed to the aqueous extract of the whole plant of E. heterophylla during biosynthesis of silver nanoparticles (Figure 2). Meanwhile, the FTIR result obtained disagrees with the works of (Govindaraju, Tamilselvan, Kiruthiga, & Singaravelu, 2010) as the FTIR spectrum of the plain S. torvum leaf extract characterized showed peaks at 1642, 1380, 1316, 1261 and 1020 cm-1. The disagreement may be due to the different plants used in the study. However, the functional groups were obtained to act as the reducing agent (Aisida et al., 2021; Nindawat & Agrawal, 2019). In addition, the TEM result (Figure 3) revealed that the CAgNPs are spherical in shapes that facilitate the delivery of drugs to the target sites, as reported in the studies of (Govindaraju et al., 2010; Saeed, Iqbal, & Ashraf, 2020). The presence of silver as revealed by the EDS spectrum (Figure 3) in the characterized CAgNPs is similar to the results obtained by (Samuggam et al., 2021; Srichaiyapol et al., 2021), in which silver was also present.

The results obtained for the rate of killing by the CAgNPs showed reductions in the populations of all the clinical bacterial isolates: E. coli strain MRE 600 (CP014197.1), Salmonella enteric subsp. Enteric serovar typhi PMO 16/13 (CP12091.1), K. pneumoniae strain HZW25 (CP025211.1) and P. fluorescens strain 2P24 (CP025542.1) between the periods of 1-7 h. These results were similar to the ones obtained in the studies of (Betts et al., 2018; Smirnova & Oktyabrsky, 2018; Srichaiyapol et al., 2021), in which there was a reduction in the population of the bacteria as the time of exposure increased. The value of 0.00log10cfu/ml was obtained at the sixth and seventh hours when E. coli strain MRE 600 (CP014197.1) was exposed to the CAgNPs, possibly showing the most significant bactericidal effect on E. coli strain MRE 600 (CP014197.1) followed by Salmonella enteric subsp. Enteric serovar typhi PMO 16/13 (CP12091.1) and bacteriostatic effect against P. fluorescens strain 2P24 (CP025542.1) and K. pneumoniae strain HZW25 (CP025211.1) Figure 5. There were increments in all the clinical bacterial isolates when exposed to the control between 1-7 h. This result is expected since the control did not contain CAgNPs.

CONCLUSION

The characterization and rate of killing of synthesized conjugated silver nanoparticles were carried out against selected clinical bacterial isolates. The CAgNPs have a bactericidal effect on E. coli strain MRE 600 (CP014197.1) and Salmonella enteric subsp. Enteric serovar typhi PMO 16/13 (CP12091.1) between 6-7 h exposures, respectively, which can be attributed to the properties of the CAgNPs. Therefore, the study suggests that the CAgNPs exhibit a strong antimicrobial activity and the potential to be developed as an alternative agent to treat bacterial infections, curb multidrug resistant bacterial infection, and further promote speedy drug delivery to the target sites.

Conflicts of interest

There is no conflict of interest among the authors.

Author contributions

The authors provide equal contributions in: collection and/or assembly of data, data analysis and interpretation and writing the article.