The ultrastructural damage of Eugenia zeyheri and Syzygium legatii acetone leaf extracts on pathogenic Escherichia coli CURRENT STATUS: UNDER REVIEW

Background: Antibiotics are commonly added to livestock feeds in sub-therapeutic doses as growth promoters and for prophylaxis against pathogenic microbes, especially those implicated in diarrhoea. While this practice has improved livestock production, it is a major cause of antimicrobial resistance in microbes against livestock and humans. This has led to the banning of prophylactic antibiotic use in animals in many countries. To compensate for this, alternatives have been sought from natural sources such as plants. While many studies have reported the antimicrobial activity of medicinal plants with potential for use as phytogenic/botanical feed additives, little information exists on their mode of action. This study is based on our earlier work and describes ultrastructural damage induced by acetone crude leaf extracts of Syzygium legatii and Eugenia zeyheri (Myrtaceae) active against diarrhoeagenic E. coli of swine origin using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescent microscopy (FM). Gas chromatography/mass spectrometry (GC-MS) was used to investigate the chemical composition of plant extracts. Results: The extracts damaged the internal and external anatomy of the cytoplasmic membrane and inner structure at a concentration of 0.04 mg/mL. Extracts also led to an increased influx of propidium iodide into treated bacterial cells suggesting compromised cellular integrity and cellular damage. Non-polar compounds such as α-amyrin, friedelan-3-one, lupeol, and β-sitosterol were abundant in the extracts. Conclusions: The extracts of S. legatii and E. zeyheri caused ultrastructural damage to E. coli cells characterized by altered external and internal morphology. These observations may assist in elucidating the mode of action of the extracts. fixed with osmium tetroxide 4 Darmstadt, in a fume hood for 30 min. Osmium tetroxide was removed and the cells were rinsed three times with the sodium phosphate buffer. Dehydration was done with increasing ethanol concentrations of 50%, 70%, 90% and 100% for 15 min each. The 100% ethanol step was repeated three times before preparation for scanning and transmission electron microscopy.


Background
Antimicrobial resistance remains a major threat to human health globally. The use of antibiotics in livestock feeds is a major cause of antibiotic resistance in humans due to the presence of residual antibiotics in edible animal products as well as environmental contamination with antibiotics derived from animal manure [1]. Due to the strong association between antibiotic resistance and the use of antimicrobials in livestock, especially as feed additives, many countries particularly in the European Union have banned use of antibiotic feed additives [2]. This has motivated the search for suitable alternatives including the use of standardized plant extracts or isolated compounds. Active and safe compounds isolated from plants can additionally serve as drug leads for therapeutic purposes [3].
Molecular biology techniques and microscopy are used to study the mechanism of action of antimicrobial compounds [4]. Electron microscopy is used in biomedical research to study the ultrastructural morphology of bacterial cells [5,6]. Scanning and transmission electron microscopy aid the visualization of images at high resolution, providing detailed information on normal or abnormal external and internal cellular morphology such as the cell membrane, cytoplasm, nucleus, organelles and cytoskeletal structures [7,8]. These methods have greater advantages over conventional light microscopy because they are able to give three-dimensional images and higher image resolutions compared to light microscopy [9].
Although many studies have reported antibacterial activity of several plant extracts, fractions and isolated compounds against a wide range of microbes, there is not much information on the ultrastructural damage.
Many Syzygium and Eugenia species have antimicrobial activity [10,11]. The fruits and leaves of Eugenia zeyheri and S. legatii are consumed by humans and animals as food [12,13]. In a recent study, acetone leaf crude extracts of Eugenia zeyheri and Syzygium legatii had excellent antimicrobial activity with MICs varying from 0.04 to 0.23 mg/ml on clinical and reference strains of E. coli. It also reduced the attachment of E. coli to intestinal cells in a Caco-2 cell adhesion assay [14].
This study reports on the morphological and ultrastructural alterations caused by crude acetone extracts of S. legatii and E. zeyheri on a diarrhoeagenic E. coli strain of swine origin using scanning and transmission electron microscopy. The effect on membrane permeability of the bacterial cells upon treatment with the extracts was also investigated with propidium iodide which is an intact membrane-impermeable fluorescent dye [15]. Gas chromatography coupled with mass spectrometry (GC-MS) was used to investigate compounds occurring in the active extracts.

Electron microscopy
There were significant external ultrastructural changes in the scanning electron microscopic investigation of treated cells compared to the control cells as early as the third hour of treatment of bacteria with both plant extracts (Fig. 1d, 1e and 2a-f). Many of the cell populations in both extracttreated bacteria appeared rough, twisted, wrinkled and misshapen. Some cells were telescoping or had invagination while others had protrusions on their surfaces. These effects were observed after 6, 12 and 24 h of exposure of the bacteria to both plant extracts.
Observations from transmission electron microscopy ( Fig. 3a-e and 4a-f) were also similar. Alterations in the cells were observed as early as the 3 rd h of extract treatment ( Fig. 3d and 3d), progressing till the 24 th h of treatment. Some cells had chromatin condensation which was packed into apoptotic-like bodies (e.g.

Fluorescent microscopy
Fluorescent microscopic results revealed an increased entry of the fluorescent dye into extracttreated bacterial cells, seen by the amount of fluorescence in treated cells compared to the untreated control ( Fig. 5d and 5e). A small amount of red fluorescence was observed in the untreated control ( Fig. 5a) which showed that most of the bacterial cells were viable and the fluorescence observed may be due to small bacteria that died naturally, while more fluorescence was observed in heat-killed bacteria (Fig. 5b).
Gas chromatography-mass spectroscopy analysis of plant extracts The gas chromatographic-mass spectrometric analysis of acetone leaf extracts of E. zeyheri (Table   1)and S. legatii (Table 2) revealed the presence of compounds such as terpenes, steroids, alkane hydrocarbons, epoxides, and saturated fatty acids. Lupeol and α-Amyrin were most abundant in E. zeyheri while friedelan-3-one was most abundant in S. legatii.

Discussion
Microscopic techniques have long been used to assess changes in cells. The changes in the bacterial morphology were determined by scanning (SEM) and transmission electron microscopy (TEM) at different times of exposure while cellular integrity after a 3 h treatment with extracts was determined by fluorescence microscopy. SEM provides insight on the effects of antibacterial agents on external morphology and surface characteristics of bacterial cells, while TEM indicates the internal architecture of normal and abnormal cells. The two techniques combined can give a useful understanding of the antibacterial mechanism of action of novel antibacterial agents [7]. The negative control acetone had no effect on the ultrastructure, but the positive control gentamicin caused many of the bacterial cells to have roughened or wrinkled surfaces with some having protrusions.  [20]. Treatment of E. coli with cinnamaldehyde caused the separation of cytoplasmic membranes from the cell wall, cell wall and cell membrane lysis, cytoplasmic content leakage, cytoplasmic content polarization, and cell distortion [21]. These findings are similar to those of the present study. The outcome of this study may therefore provide further insights into innovative alternative methods of bacterial management through disruption of the pathogen integrity.
The integrity of the bacterial membrane following exposure to E. zeyheri and S. legatii was assessed using a fluorescent dye, propidium iodide. This dye is known to intercalate with bases of deoxyribonucleic acid (DNA) to fluoresce. Propidium iodide can enter only a compromised bacterial cell membrane to bind with DNA as an intact cell membrane will exclude it.
Increased fluorescence was also detected in gentamicin-treated cells, indicating that it also affected the cell membrane. The primary effect of gentamicin on bacteria is to bind on the 30S subunit of bacterial ribosome{Etebu, 2016 #2}, the structural damage observed is therefore a secondary effect.
The high fluorescence observed from treatment of bacteria with the extract of E. zeyheri and S. legatii indicated damage to the integrity of the bacterial cell membrane. In a similar study, the aqueous extract of Cassia alata caused a high number of cell deaths in Streptococcus epidermidis and Pseudomonas aeruginosa based on fluorescence microscopy [22].
The fluorescence results support the observations in the electron microscopy study. Cell abnormalities observed in the electron microscopy results such as abnormal cellular integrity, and/or partial or complete loss of cytoplasmic contents may be responsible for the highly permeable state of the cells to PI. It appeared that the compounds present in the extracts were able to penetrate the peptidoglycan layer of the bacterial cell into the cell membrane to exert the antibacterial effects.
Possible targets for the compounds may be present on this layer [23]. More studies are needed to confirm these observations. In a previous study, lupeol, isolated from Curtisia dentata leaves had antibacterial activity against E. coli (ATCC 25922) with an MIC of 250 [24]. In another report, α-amyrin had an MIC of 64 µg/mL against Staphylococcus aureus (ATCC 43300) [25]. It may be worthwhile to isolate and characterize the bioactive compounds from the plant extracts from this study and determine their antibacterial activity and safety which may serve as new antibacterial drugs.

Conclusion
The extracts of S. legatii and E. zeyheri caused morphological and ultrastructural damage to E. coli when exposed to inhibitory concentrations of the extracts. These effects were characterized by disruptions and disfigurations in the cell shape and surface cell wall formation. The extracts affected the membrane integrity of the bacterial cells shown by increased fluorescence. We conclude that the plant extracts have potential for therapeutic use. Further work may include exploring the molecular mechanisms and precise cellular target(s) responsible for the effects seen in this study. Model A10 mill, the dried leaves were ground to a fine powder, weighed and stored in at room temperature in closed jam jars [26].

Extraction
Two grams of dried powdered plant material was extracted with 20 mL of acetone technical grade, Merck) in 50 mL centrifuge tubes. Acetone is widely considered a solvent of choice because it can extract compounds with a wide range of polarities, it is relatively easy to remove from extracts and it is non-toxic to bioassays systems [27]. The tube containing the mixture was vigorously shaken and sonicated for 20 min followed by centrifugation for 10 min at 4000 X g. The supernatant was then filtered using a Whatman No. 1 filter paper into a pre-weighed glass container and then dried under a cold stream of air in a fume hood at room temperature to obtain a dried extract. The dried extracts for were dissolved in the required volume of acetone for the bioassays..

Bacterial strain
An enterotoxigenic E. coli (possessing the STA and F6 virulence genes) isolated from a diarrhoeic piglet was obtained from the Department of Veterinary Tropical Diseases, Faculty of Veterinary Sciences, University of Pretoria. The organism was maintained on Tryptic Soy Agar (TSA, Oxoid) at Preparation of E. coli culture for electron microscopy Escherichia coli was grown in TSA for 18 h after which a single colony was inoculated into Tryptic Soy Broth aseptically and incubated at 37 o C on a shaker for 18 h. After this, an inoculum equivalent to a McFarland No 1 standard (3.6 × 10 8 cfu/mL) was prepared from the 18 h culture. Prior to this, TSA was prepared, and 5 mL of the molten agar was gently poured into 35 mm diameter sterile tissue culture plates to form a smooth and evenly spread surface and allowed to solidify in a sterile environment. The 35 mm agar plates were inoculated with the appropriately adjusted E. coli suspension which were spread evenly on the agar surface with a sterile glass spreader. The plates were then incubated at 35 0 C for 12 h under aerobic conditions. The plates were divided into two groups. Plates in group one was flooded with 100 µl of E. zeyheri extract while those in group two were flooded with the extract of S. legatii, bothat 0.04 mg/mL in acetone. This concentration represented the minimum inhibitory concentration of the extracts on the test bacterium in our previous study. One plate was flooded with 100 µl of 50% acetone to represent the solvent control.
One plate also served as the untreated control, while another was treated with 100 µl gentamicin (1 µg/mL) as positive control. The plates were then incubated under aerobic conditions at 37 0 C. After 0, 3, 6, 12, and 24 h, separate plates were removed from the incubator and flooded with 1 mL of 2.5% glutaraldehyde in 0.075 M phosphate buffer solution (pH 7.4) to fix the samples for 60 min. The bacterial biofilms were then collected from each plate using sterile loops and transferred into 2 mL microcentrifuge tubes containing 1.5 mL of 0.5% glutaraldehyde in order to fix the cells for 1 h.
Glutaraldehyde was removed with a pipette and the cells were washed thrice with 0.075 M sodium buffer for 10 min each. The samples were then fixed with osmium tetroxide (OsO 4 , Merck, Darmstadt, Germany) in a fume hood for 30 min. Osmium tetroxide was removed and the cells were rinsed three times with the sodium phosphate buffer. Dehydration was done with increasing ethanol concentrations of 50%, 70%, 90% and 100% for 15 min each. The 100% ethanol step was repeated three times before preparation for scanning and transmission electron microscopy.
Scanning electron microscopy (SEM) sample processing Following the last 100% ethanol dehydration from the step above, hexamethyldisilazane (HMDS) was added at 50% in ethanol for 30 min. Hexamethyldisilazane/ethanol was replaced with two changes of pure HMDS for 1 h each. A small droplet (0.05 mL) containing sample was placed on highly polished carbon discs and left open in a fume hood to dry overnight. These carbon discs were stuck using double-sided carbon tape onto aluminium stubs. Samples were made conductive by exposure to ruthenium tetroxide (RuO 4 ) for 45 min [28]. Samples were then viewed with a Zeiss Ultra Plus Field Emission Gun Scanning Electron Microscope (FEGSEM) at the Electron Microscope Unit of the University of Pretoria.

Transmission electron microscopy (TEM) sample processing
After the third dehydration step in 100%, ethanol was removed and replaced with propylene for 2 h.
The propylene oxide was replaced with an Epon type epoxy resin (TAAB 812) and infiltrated for 5 h.