Combatting Drug-Resistant Clostridium perfringens Type B: In vitro effect of Eucalyptus globulus Essential Oil Fractions
Zian Asif1, Shahan Azeem1*, Aftab Ahmad Anjum1 and Muhammad Muddassir Ali2
1Institute of Microbiology, Faculty of Veterinary Science, University of Veterinary and Animal Sciences, Lahore, 54000, Pakistan
2Institute of Biochemistry and Biotechnology, Faculty of Bio-Sciences, University of Veterinary and Animal Sciences, Lahore, 54000, Pakistan
ABSTRACT
The development of antimicrobial resistance in Clostridium perfringens could pose a significant challenge to public health, necessitating the exploration of alternative treatment options such as essential oils. Essential oils have promising antimicrobial properties that need further exploration. Plant essential oils including Eucalyptus globulus essential oil has gained attention for its diverse therapeutic attributes. The present study aimed to evaluate the efficacy of cinnamon, clove, coriander, and Eucalyptus (E. globulus) essential oils. The activity of E. globulus essential oils fractions was evaluated in laboratory settings against 3 antibiotic-resistant C. perfringens type B isolates recovered from local sheep. The isolates were confirmed by polymerase chain reaction (PCR) targeting 16S rRNA gene. The Kirby-Bauer disc diffusion method was used to ascertain drug resistance. The C. perfringens isolates were resistant to ciprofloxacin and pefloxacin. Of the four tested essential oils, E. globulus exhibited the highest zone of inhibition (14 mm), followed by coriander oil (1 mm), while the Cinnamon and Clove essential oils did not show any zone of inhibition. The potential of nine distinct fractions of E. globulus essential oil to inhibit growth of drug-resistant C. perfringens was evaluated. The minimum inhibitory concentration (MIC) for the E. globulus oil fractions was 4-26 g/ml against antibiotic-resistant C. perfringens. The n-hexane+chloroform fraction was found to be the most effective with an MIC of 4.833±2.09 µg/ml suggesting in vitro antibacterial activity against drug-resistant C. perfringens.
Article Information
Received 10 November 2021
Revised 20 April 2025
Accepted 09 May 2025
Available online 23 June 2025
(early access)
Published 12 March 2026
Authors’ Contribution
ZA, AAA and SA contributed to the study’s conception and design. ZA conducted the experiments, collected data and wrote the first draft of the manuscript. SA supervised the study, analyzed data, critically reviewed the manuscript. AAA provided the chemical, reagents, and laboratory facility for the research work. SA and MMA critically reviewed and revised the manuscript. All authors approved the final manuscript.
Key words
Minimum inhibitory concentration, Polymerase chain reaction, Antimicrobial resistance, Gas chromatography, Mass spectrometry
DOI: https://dx.doi.org/10.17582/journal.pjz/20211110101157
* Corresponding author: [email protected]
0030-9923/2026/0003-1025 $ 9.00/0
Copyright 2026 by the authors. Licensee Zoological Society of Pakistan.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Introduction
Clostridium perfringens is a Gram-positive, spore-forming, toxigenic, anaerobic, rod-shaped bacterium that is ubiquitous in nature (Johnston et al., 2022). It is a fast-growing bacterium found in soil, manure, rotting vegetation, and environments contaminated with feces (Bush and Vazquez-Pertejo, 2023). While it is commonly found in human and animal intestines as a part of normal gut microbiota, it has the potential to become pathogenic when the gut microbiome is perturbed due to stress caused by hunger, abrupt changes in diet, or the therapeutic use of antibiotics (Redondo et al., 2015; Mohiuddin et al., 2020). C. perfringens is most commonly associated with food-borne illnesses owing to its ability to produce enterotoxins. Based on toxins, C. perfringens can be categorized into seven toxinotypes: A through G. The bacterium produces specific toxins, such as alpha, beta, epsilon, iota, enterotoxin, beta-2, and NetB (Fernandez-Miyakawa et al., 2007).
C. perfringens Type B causes enteric diseases in various animals, including cattle, goats, and sheep (Uzal et al., 2018). In sheep, C. perfringens is the main cause of necro-hemorrhagic enteritis (Uzal et al., 2018). C. perfringens type B is the predominant isolate in sheep, followed by type A and type D (Nayel et al., 2013). C. perfringens type A and D are commonly isolated from clinically healthy sheep (Nayel et al., 2013). C. perfringens Type B can cause a range of diseases in sheep, including neonatal lamb dysentery, a significant cause of mortality in young lambs. Recently a case of fatal neonatal lamb gastroenteritis was reported in New Zealand (Munday et al., 2020). The lamb was found dead at day one of age, and a post-mortem examination revealed severe mucosal necrosis and ulceration in the small intestine, consistent with Clostridial enteritis. The isolation and characterization confirmed the presence of C. perfringens type B (Munday et al., 2020). Another study described a case of suspected neurotoxicity in a tiger (Panthera tigris) caused by C. perfringens type B. The tiger, a 5-years old male, was presented to a veterinary clinic with neurological signs including ataxia, tremors, and seizures. A diagnosis of suspected neurotoxicity was made based on the clinical signs and the presence of C. perfringens type B in the tiger’s feces. The tiger was treated with antibiotics and supportive care but ultimately died due to the severity of neurological signs. This case highlights the potential of C. perfringens type B to cause neurotoxicity in tigers, and the importance of considering this in the differential diagnosis in cases of neurological signs in captive tigers (Zeira et al., 2012). C. perfringens type B has also been found associated with multiple sclerosis in humans (Rumah et al., 2013).
Several antibiotics are used to treat C. perfringens infections in humans and animals. However, antimicrobial resistance to many of the commonly used antibiotics has been documented which renders treatment ineffective (Khanna, 2008; Beres et al., 2023). Plant-derived essential oils possess antibacterial properties and can be used as an alternative to antibiotics (Tariq et al., 2019; Liu et al., 2020). These essential oils are composed of volatile/phenolic chemicals and aromatic compounds (Dhifi et al., 2016). The phenolic compounds disrupt the proton motive force, cytoplasmic membrane, active transport, and electron flux, causing the coagulation of bacterial cells (Divya et al., 2014). Additionally, phenolic compounds lead to the breakdown of the lipid connections linking the protein layers, and the coagulation of cellular components in the cytoplasm (Trombetta et al., 2005). Essential oils function in a manner that is comparable to antibiotics target sites suggesting essential oils may substitute antibiotics (Yap et al., 2014). Essential oils from Eucalyptus globulus have antimicrobial and anticoccidial properties (Javed et al., 2023). The essential oil of various Eucalyptus species have antioxidant and antimicrobial properties (Almas et al., 2021). Eucalyptus oil’s antimicrobial activity could be attributed to the presence of 1,8-cineole, α-pinene, β-pinene, and limonene. Additionally, isoborneol, a bicyclic monoterpene alcohol, present in E. globulus essential oil, disrupts the bacterial cell membrane and inhibits its essential enzymes hindering bacterial growth (Merghni et al., 2023). The present study was designed to evaluate the ability of cinnamon clove, coriander, and Eucalyptus (E. globulus) essential oils against antibiotic-resistant C. perfringens type B in laboratory settings.
Materials and Methods
Characterization of Clostridium perfringens isolates
Three isolates of Clostridium perfringens type B were obtained from the Quality Operations Laboratory, Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan. The samples originated from sheep in the Lahore and Kasur districts of the Punjab Province of Pakistan. Perfringens agar base medium was used to revive the bacterium. The agar plates were incubated for 24 h at a temperature between 37 to 40ºC in anaerobic conditions. Any black and round colonies observed on the Perfringens agar base medium plates were suspected to be C. perfringens. The suspected colonies were subjected to Gram staining, microscopy, and biochemical testing. The colonies presumptively positive for C. perfringens were subjected to PCR. The bacterial DNA was extracted using a commercial DNA extraction kit (GeneAll, Seoul, Korea). Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) was used to estimate the concentration of the extracted DNA. A multiplex PCR targeting alpha, beta, and epsilon toxins genes of C. perfringens was set up using published primer information (van Asten et al., 2009). The PCR reaction mixture was composed of 12.5 µL of 2X PCR master mix (ThermoFisher Scientific, Waltham, MA, USA), 1.5 µL each of forward and reverse primers (10 pmol) for each of the three toxin genes, 2 µL of DNA template, and 7.5 µL of nuclease-free water. PCR cycling conditions were as follows: polymerase activation at 94°C for 10 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min, and a final extension at 72°C for 10 min. The PCR products were analyzed using 1.5% agarose gel containing 0.5 µg/mL ethidium bromide. The gel was visualized using a gel documentation system (BioRad, Hercules, CA, USA).
Antibiotic resistance patterns
The PCR-confirmed C. perfringens isolates (n=3) were tested for their susceptibility to various antibiotics. The Kirby-Bauer disc diffusion method was used following Clinical and Laboratory Standards Institute (CLSI, 2020) guidelines. The bacterial broth suspension was adjusted to 0.5 McFarland units by measuring the optical density at 630 nm using a spectrophotometer (Neogen, Lansing, MI, USA). The inoculum was cultured on nutrient agar plates using the spread plate method. Antibiotic discs containing nalidixic acid, meropenem, cefepime, penicillin, nitrofurantoin, norfloxacin, pefloxacin, cefotaxime, linezolid, ciprofloxacin, vancomycin, amoxicillin, ceftazidime, levofloxacin, and ceftriaxone were placed on the agar plates and gently pressed using sterile forceps. After 24 h of incubation at 37°C, the plates were examined for bacterial growth and a clear zone of inhibition (ZOI) indicating antibiotic sensitivity was noted. The diameter of the ZOI was measured to the nearest millimetre using a ruler and compared to the CLSI (2022) guidelines to determine whether the bacteria were sensitive to antibiotics (CLSI, 2022).
Determination of the activity of Eucalyptus globulus essential oil fractions
The study employed the broth microdilution method to evaluate the minimum inhibitory concentration (MIC) of E. globulus essential oil. A commercial product containing E. globulus essential oil (Marhaba Laboratories Private Limited, Lahore, Pakistan) was procured from the local market. Column chromatography was used to fractionate the essential oils according to increasing solvent polarity, using silica gel as the stationary phase, as previously described (Moein et al., 2015; Kaur et al., 2019). Different solvents, including n-hexane (14.15 µg/ml), chloroform (6.96 µg/ml), ethyl acetate (18.64 µg/ml), methanol (19.16 µg/ml), and acetonitrile (26.87 µg/ml), along with their combinations, were used to elute the essential oil.
The MIC determinations were made by using 96-well microtiter plates. Serial dilution of essential oil was prepared in liquid Perfringens agar base (PAB) medium. Test bacterial strains were suspended in the medium. McFarland standard 0.5 units was used to adjust the cell densities through a spectrophotometric method. A 96-well microtiter plate was prepared by adding 0.1 mL PAB broth to each well. One hundred microliters of test extracts were added in the first well and a two-fold dilution of oil extract was made by using a micropipette up to the 10th well. The microtiter plate was inoculated with approximately 0.1 mL of the bacterial inoculum up to the 11th well and incubated anaerobically at 37°C for 24 to 48 h. Optical density was measured at 0 and 24 h. The 11th well served as the positive control, while the 12th well was the negative control.
Gas chromatography and mass spectrometry
To analyse the samples using gas chromatography and mass spectrometry (GCMS), a CARBOWAX capillary column was utilized along with helium as the carrier gas. An injector was heated to 260 ºC, and E. globulus essential oil’s n-hexane and n-hexane + chloroform solvent fractions were injected at a rate of 1 µL/min (Kaur et al., 2019). The identification of active compounds in the test samples was accomplished by comparing their retention time to that of standard compounds.
Statistical analysis
Data based on the zones of inhibition and MIC values of oil fractions were analyzed using one-way analysis of variance (ANOVA) followed by post hoc Duncan’s multiple range test. Statistical Package for Social Sciences (SPSS) version 20.0 (IBM, Armonk, NY, USA) was used for statistical analysis. The level of significance was set at 0.05.
Results
The Clostridium perfringens type B isolates, formed characteristic black, round colonies due to sulphite reduction, when cultured on Perfringens agar base medium under anaerobic conditions at 37–40°C for 24 h. Gram staining revealed Gram-positive, rod-shaped bacilli with blunt ends. Microscopic examination showed non-motile, short chains or single cells with a thick peptidoglycan layer.
Characterization of isolates
The PCR targeting alpha (324 bp), beta 158 (192 bp), and epsilon (376 bp) toxin genes of C. perfringens indicated the presence of suspect bacterium (Fig. 1).
Column L shows the 100 bp ladder. Column A is the Alpha gene of 324 bp. Column B is the Beta gene of 192 bp. Column C shows the bands of the Epsilon gene at 376 bp. Columns A-C, D-F, and G-I correspond to Alpha, Beta, and Epsilon genes, respectively of Clostridium perfringens Type B isolates CZ-01, CZ-02, and CZ-03, respectively.
Table I. Evaluation of antibiotic activity against Clostridium perfringens type B.
|
Antibiotics |
Concentration |
CZ-01 (mm) |
CZ-02 (mm) |
CZ-03 (mm) |
Mean ±S.D |
Sensitive/ Resistant |
|
Nalidixic acid |
30µg |
18 |
20 |
23 |
20±2.51ab |
Sensitive |
|
Meropenum |
10 µg |
50 |
58 |
60 |
56±5.29f |
Sensitive |
|
Cefepime |
30 µg |
37 |
35 |
46 |
39±5.85c |
Sensitive |
|
Penicillin |
10 µg |
78 |
69 |
78 |
75±5.1g |
Sensitive |
|
Nitrofurentoin |
300 µg |
39 |
42 |
44 |
41.66±2.51cd |
Sensitive |
|
Norfloxacin |
10 µg |
21 |
20 |
20 |
20±0.57ab |
Sensitive |
|
Pefloxacin |
5mcg |
12 |
13 |
20 |
15±4.35a |
Resistant |
|
Cefotaxime |
30 µg |
52 |
50 |
57 |
53±3.60ef |
Sensitive |
|
Linezolid |
30 µg |
35 |
43 |
50 |
42.66±7.50cd |
Sensitive |
|
Ciprofloxacin |
5 µg |
23 |
25 |
28 |
25±2.51b |
Resistant |
|
Vancomycin |
30 µg |
45 |
48 |
50 |
47.66±2.51de |
Sensitive |
|
Amoxicillin |
30 µg |
54 |
59 |
60 |
57.66±3.21f |
Sensitive |
|
Ceftazidime |
30 µg |
43 |
46 |
48 |
45.66±2.51abc |
Sensitive |
|
Levofloxacin |
5 µg |
24 |
22 |
30 |
25±4.16b |
Sensitive |
|
Ceftriaxone |
30 µg |
48 |
59 |
60 |
55.66±6.65f |
Sensitive |
*Means with different superscripts differ significantly.
Antibiotic resistance profile and well diffusion assay
The highest zone of inhibition (ZOI) was demonstrated by penicillin (75±5.1 mm) followed by amoxicillin (57.66±3.21 mm). The least ZOI was noted for pefloxacin (15±4.35 mm) followed by ciprofloxacin (25±2.51 mm). The resistance patterns against various antibiotics are presented in Table I.
The well diffusion assay demonstrated a distinct zone of inhibition (14 mm) around the Eucalyptus globulus in comparison to the cinnamon oil, clove oil, and coriander oil (Fig. 2).
Activity of E. globulus oil fractions
Nine different fractions of the E. globulus oil were evaluated in this study. The n-hexane+chloroform fraction demonstrated the least MIC (4.833±2.09 µg/ml) followed by the chloroform fraction (6.96±0.00 µg/ml). These fractions were further processed for GCMS analysis. The highest MIC was noted for the acetonitrile fraction (26.87±0.00 µg/ml) followed by the chloroform + ethyl acetate (23.22±8.04 µg/ml) fraction. The MICs of various fractions are presented in Table II.
Gas chromatography and mass spectrometry analysis
For component analysis and identification of the concentration of an active chemical in the n-hexane+ chloroform fraction, gas chromatography and mass spectrometry (GCMS) were performed. The identification and quantification of components by GCMS were based on comparing the mass spectrum of the E. globulus essential oil with the spectra reported previously. The concentrations of the active components found in the essential oil fractions after GCMS are presented in Table III.
Table II. Mean MIC Values of Eucalyptus globulus oil fractions.
|
Mean MIC values of Eucalyptus globulus oil fractions |
||||
|
Solvent fraction |
MIC values |
Mean ±S.D |
||
|
Iso-01 |
Iso-02 |
Iso-03 |
||
|
n-hexane |
14.15 |
14.15 |
14.15 |
14.10±0.00ab |
|
n-hexane+chloroform |
3.625 |
7.25 |
3.625 |
4.833±2.09a |
|
Chloroform |
6.96 |
6.96 |
6.96 |
6.96±0.00a |
|
Chloroform+ ethyl acetate |
27.87 |
27.87 |
13.93 |
23.22±8.04bc |
|
Ethyl acetate |
27.96 |
13.98 |
13.98 |
18.64±8.07bc |
|
Ethyl acetate+methanol |
14 |
28 |
14 |
18.66±8.08bc |
|
Methanol |
28.75 |
14.37 |
14.37 |
19.16±8.30bc |
|
Methanol+acetonitrile |
7.1 |
14.37 |
14.37 |
19.16±8.30bc |
|
Acetonitrile |
26.87 |
26.87 |
26.87 |
26.87±0.00c |
*Means with different superscripts differ significantly. The inhibitory effect observed in the study is primarily attributed to the active compounds present in the essential oil of Eucalyptus globulus, not the solvents used for fractionation. The main components, such as Bicyclo(2,2,1)heptan-2-ol,1,7,7-trimethyl, Benzene, Isoborneol, and Bicyclo(2,2,1)heptan-2-ol, have been identified through GCMS analysis and are known for their antimicrobial properties. The effectiveness of the Eucalyptus globulus essential oil against antibiotic-resistant Clostridium perfringens is mainly due to these bioactive components present in the oil, indicating that the inhibitory effect is a result of the essential oil itself.
The bicyclo(2.2.1)heptan-2-ol, 1,7,7-trimethyl fraction had the highest concentration (14.4%), followed by cyclohexane (13%), benzene (9.5%), and o-cymene (6.4%). The lowest concentration was noted for camphor (0.39%) followed by exo-2,7,7-trimethylbicyclo (2.2.1) heptan-2-ol (0.62%), and Terpinene (0.62%) fractions.
Discussion
The emergence of antimicrobial resistance in Clostridium perfringens has highlighted the need for alternative treatment strategies. The present study was conducted to evaluate the effectiveness of cinnamon, clove, coriander, and eucalyptus (E. globulus) essential oils against antibiotic-resistant C. perfringens type B in laboratory settings. The study results indicated that E. globulus is effective against C. perfringens with an MIC of 4.833±2.09 µg/ml. The study highlights the potential antimicrobial of E. globulus essential oil against drug-resistant C. perfringens type B. By focusing on the most effective fraction, n-hexane+chloroform, we present in vitro evidence that E. globulus essential oil components can be effective against antimicrobial-resistant C. perfringens type B.
Table III. Concentrations and retention time of the active compounds of Eucalyptus globulus essential oil fractions.
|
Name |
Retention time |
Percentage concentration |
|
Cyclohexane |
6.1 |
13 |
|
Bicyclo(2.2.1)heptane, 2,2-dimethyl-3-methylene |
13.6 |
4.5 |
|
7-Oxabicyclo(2.2.1)heptane, |
15 |
0.87 |
|
1,3,5-cycloheptatriene |
15.1 |
0.70 |
|
o-cymene |
15.3 |
6.4 |
|
Cyclohexanol |
15.4 |
1.48 |
|
Eucalyptol |
15.54 |
5.71 |
|
Terpinene |
16 |
0.62 |
|
Benzene |
16.68 |
3.6 |
|
Bicyclo(2.2.1)heptane |
16.76 |
1.7 |
|
Bicyclo(2.2.1)heptan-2-one |
17.0 |
4.9 |
|
Bicyclo (2.2.1)heptan-2-ol |
17.3 |
8.7 |
|
exo-2,7,7- trimethylbicyclo (2.2.1)heptan-2-ol |
17.8 |
0.62 |
|
Camphor |
18 |
0.39 |
|
Isoborneol |
18.28 |
5.35 |
|
Bicyclo(2.2.1)heptan-2-ol, 1,7,7-trimethyl |
18.42 |
14.4 |
|
Benzene |
18.59 |
9.5 |
|
L-α-terpineol |
18.7 |
1.5 |
|
Acetaldehyde |
19 |
0.72 |
|
Bicyclo (3.1.0) hexane |
19.15 |
3.9 |
|
Cinnamaldehyde |
20.1 |
1 |
|
Octanal |
21.2 |
0.9 |
|
(1,1’-Bicyclopropyl)-2-octanoic acid |
21.4 |
1.7 |
The antimicrobial resistance findings of the present study are consistent with Slavić et al. (2011) who investigated the minimal inhibitory concentrations (MICs) of 12 antimicrobials against C. perfringens isolates of cattle, swine, turkeys, and chickens -origin and reported reduced susceptibility to bacitracin, clindamycin, erythromycin, florfenicol, and tetracycline (Slavić et al., 2011). The present study’s results confirm the development of antibiotic resistance in C. perfringens. The development of antimicrobial resistance in C. perfringens, consistent across both studies, have implications for both animal and human health. In livestock, reduced susceptibility to key antibiotics jeopardizes the effectiveness of treatments, potentially leading to more severe infections and prolonged recovery periods. Moreover, the zoonotic potential of C. perfringens raises alarms, as resistant strains can be transmitted from animals to humans highlighting the risk of antibiotic-resistant infections in both populations (Bendary et al., 2022).
The present study results of antimicrobial activity of E. globulus essential oil corroborate Ávila et al. (2023) findings who have reported antibacterial activity of aromatic plant ethanolic extracts against Clostridium spp. (Ávila et al., 2023). The present study also confirms the results of a Chinese study suggesting the antimicrobial activity of globulol, a compound found in E. globulus Labill plant. The study evaluated the antimicrobial activity of globulol against a range of microorganisms, including bacteria and fungi. The results indicated that globulol could be a main antimicrobial compound in the ethanol extract of E. globulus (Tan et al., 2008). The present study’s findings are also consistent with those of Ullah et al. (2021) who reported the antimicrobial activity of Eucalyptus essential oil against C. perfringens (Ullah et al., 2021).
In the present study, the n-hexane+chloroform fraction of E. globulus oil was found to have the least MIC (highest antibacterial activity) value of 4.833±2.09 µg/ml. The GCMS of the n-hexane+chloroform fraction revealed that bicyclo (2.2.1) heptan-2-ol, 1,7,7-trimethyl had the highest concentration (14.4%), followed by cyclohexane (13%), benzene (9.5%), and o-cymene (6.4%). A similar study using GCMS analysis revealed that the main chemical compounds in E. globulus were oxygenated monoterpenes (78.58%), 1,8-cineole (55.29%), spathulenol (7.44%), and a-terpineol (5.46%) (Harkat-Madouri et al., 2015). Findings of another GCMS analysis of E. globulus essential oil fraction revealed following key compounds: 1.8-cineole (22.35%), limonene, (7.01%), solanol (6.05%), p-pinene (5.20%), transverbenol (4.02%), terpinen-4-ol (3.10%), aristolene (2.35%), terpinyl acetate (2.10%), isosativene (1.85%), sabinene (1.49%), (alpha)-myrcene (1.15%), and a-terpineol (1.10%) (Jerbi et al., 2017). The active compounds suggested by GCMS in the present study were different from those reported by (Harkat-Madouri et al., 2015; Čmiková et al., 2023). The observed differences in the active compounds between studies can be attributed to variations in the plant’s chemical composition owing to geographical differences and the use of different solvents for GCMS analysis (Almas et al., 2019). Environmental factors in different geographical regions influence the plant’s natural chemical makeup, while varying solvents selectively extract different compounds from the essential oil, thus leading to variations in the identified components (Harkat-Madouri et al., 2015; Jerbi et al., 2017).
The fraction n-hexane+chloroform demonstrated the lowest MIC against the tested isolate: 4.833±2.09µg/ml. The MIC results suggest that the E. globulus oil can be used as an alternative to antibacterial agents. The major components of E. globulus, bicyclo (2,2,1) heptan-2-ol,1,7,7-trimethyl, benzene, isoborneol, bicyclo (2,2,1) heptan-2-ol, contribute to the antimicrobial activity corroborating Ali et al. (2023, who have also reported the antimicrobial activity of this component ().
The mechanism of action of bicyclo (2,2,1) heptan-2-ol,1,7,7-trimethyl, benzene, isoborneol, and bicyclo(2,2,1) heptan-2-ol, as an antibacterial agent in E. globulus essential oil, is likely multifaceted. Bicyclo (2,2,1) heptan-2-ol, also known as α-terpineol, disrupts bacterial cell membrane, increases its permeability, and inhibits its protein synthesis, impeding bacterial growth. Benzene, as an aromatic hydrocarbon, may contribute to overall antimicrobial activity, although its exact mechanism of action remains unclear. Isoborneol present in E. globulus essential oil, disrupts the bacterial cell membrane and inhibits essential enzymes, hindering bacterial growth (Rajput et al., 2023).
Some of the limitations of the present study include, no assessment of the cytotoxicity of E. globulus essential oil fractions. Moreover, the study did not determine the activity of E. globulus essential oil against extensive drug-resistant strain of C. perfringens. These aspects require further investigation. Besides, therapeutic implications of the current study could be clarified by additional in vivo investigations on the effectiveness of E. globulus essential oil in treating C. perfringens type B infections in humans and animals (Assaggaf et al., 2022).
Future studies could evaluate the active ingredient of Eucalyptus globulus essential oil, bicyclo (2,2,1) heptan-2-ol,1,7,7-trimethyl, in the n-hexane+chloform fraction, for resistance modulation studies (Zielińska-Błajet and Feder-Kubis, 2020). Moreover, Eucalyptus globulus essential oils should be evaluated for oral administration in humans and poultry (Ahlem et al., 2009; Ullah et al., 2021) to prevent or control bacterial infections.
Conclusions
The present study indicates that Eucalyptus globulus is a promising antibacterial agent, and its n hexane+chloform fraction have in vitro antibacterial activity against C. perfringens type B. E. globulus oil can be used as an alternative to antibiotics.
Declarations
Acknowledgement
The authors are grateful to Ms. Rabia Manzoor for her extraordinary help in executing laboratory experiments.
Funding
The authors would like to acknowledge the funding support provided by the Higher Education Commission of Pakistan (Project No. TDF-02-028).
Statement of conflict of interest
The authors have declared that there is no conflict of interest.
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