Eucalyptus camaldulensis for combating the Multi Drug Resistance Bacterial Strains (MDRS)

Eucalyptus camaldulensis para combater as cepas bacterianas multirresistentes a medicamentos (MDRS)

Abstract

Antibiotic resistance in bacteria is the primary challenge for health worldwide. The widespread of food poisoning due to use of stored food items increasing day by day. The present study was designed to research the protective effect of Eucalyptus camaldulensis methanolic extract again selected strains of bacteria (Escherichia coli, Bacillus cereus, Bacillus subtilis and Staphylococcus aureus). The treatment was used for several concentrations and then characterized on the basis of temperature and pH variations. Results were observed by MIC (minimum inhibitory concentration) against these strains. It was noted that maximum inhibition of E. camaldulensis against E.coli (5.6mm) was observed at 250 mg/mL while it was 3.6mm against B. cereus at 200 mg/mL, Staph. aureus showed maximum (3.1mm) zone at 250 mg/mL. at variable temperature of E. camaldulensis extract, it was observed that MIC of B. cereus was 6.4mm at 80 °C. For other strains the results revealed that the maximum zone against B. subtilis was 5.7mm at 121 °C and for Staph. aureus it was 6.6mm at 80 °C. By observing the results by changing pH it was observed that MIC produced against B. cereus was 6.2mm at 7pH, against B. subtilis zone of inhibition was 7.2mm at 5pH, for E.coli it was 5.4mm at 3pH. Means of all the variable results of different (concentration, pH and temperature) were compared by using one way ANOVA. The current study suggested that the methanolic extract of E. camaldulensis was found effective in control of antibiotic resistant strains and this study strengthens the fact of using herbal solutions for control of antibiotic resistant bacterial infections.

Keywords:
E. camaldulensis; methanolic extract; Escherichia coli; Bacillus cereus; Bacillus subtilis; Staphylococcus aureus

Resumo

A resistência bacteriana a antibióticos é o principal desafio para a saúde em todo o mundo. A disseminação da intoxicação alimentar devido ao uso de alimentos armazenados aumenta a cada dia. O presente estudo foi elaborado para pesquisar o efeito protetor do extrato metanólico de Eucalyptus camaldulensis em cepas selecionadas de bactérias (Escherichia coli, Bacillus cereus, Bacillus subtilis e Staphylococcus aureus). O tratamento foi aplicado em várias concentrações e então caracterizado com base nas variações de temperatura e pH. Os resultados foram observados por MIC (concentração inibitória mínima) contra essas cepas. Foi notado que a inibição máxima de E. camaldulensis contra E. coli (5,6 mm) ocorreu na concentração de 250 mg/mL, enquanto foi de 3,6 mm contra B. cereus a 200 mg/mL, e Staph. aureus apresentou zona máxima (3,1 mm) a 250 mg/mL. Em temperatura variável do extrato de E. camaldulensis, observou-se que a MIC de B. cereus foi de 6,4 mm a 80 °C. Para outras cepas, os resultados revelaram que a zona máxima contra B. subtilis foi de 5,7 mm a 121 °C, e para Staph. aureus foi de 6,6 mm a 80 °C. Observando os resultados pela alteração do pH, observou-se que a MIC produzida contra B. cereus foi de 6,2 mm a 7 pH, contra B. subtilis a zona de inibição foi de 7,2 mm a 5 pH, para E. coli foi de 5,4 mm a 3 pH. As médias de todos os resultados variáveis ​​ (concentração, pH e temperatura) foram comparadas usando ANOVA unidirecional. O presente estudo sugeriu que o extrato metanólico de E. camaldulensis foi considerado eficaz no controle de cepas resistentes a antibióticos, e este estudo reforça o uso de soluções fitoterápicas para o controle de infecções bacterianas resistentes a antibióticos.

Palavras-chave:
E. camaldulensis; extrato metanólico; Escherichia coli; Bacillus cereus; Bacillus subtilis; Staphylococcus aureus

1. Introduction

The most important treatment for harmful bacteria is antibiotics. Overuse of antibiotics without proper medical justification has led to the rise of drug-resistant bacteria. Natural antibacterial properties of herbs are also a reason because of which they are often used in modern medicine (Hiwandika et al., 2021). There has been a steady rise in the percentage of bacteria that are resistant to common antibiotics. Misuse of these medicines has been linked to the development of drug-resistant bacteria and yeasts, include enterobacteria that produce extended-spectrum beta-lactamases and are carbapenem-resistant as well as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, drug-resistant Streptococcus pneumoniae, and Pseudomonas and Aeruginosa. (Aslam et al., 2018).

Researchers have spent a lot of time over the years trying to find ways to slow bacterial growth using plant extracts. There are many different drugs may be found in plants. Phytochemicals, which are produced during the plant's secondary metabolism, are responsible for the antibacterial characteristics. (Manandhar et al., 2019).

Eucalyptus camaldulensis bush has a significant role in the field of medicine. The River Red Gum, or Eucalyptus camaldulensis, is a member of the Myrtaceae family of plants. Around 3,800 members of 140 different genera populate the tropics and subtropics of every continent. The genus Eucalyptus was first recognized and named in 1788 by French botanist Héritier. Since the flowers of many species of Eucalyptus are shielded by an operculum, the generic name “eucalyptus” comes from the Greek words “eu” (well) and “kalyptos” (covered). It originated in Australia and Tasmania, but because to its tolerance and quick growth, it has spread over the world and been successfully imported. Consequently, Eucalyptus has become one of the most widely planted genera and plantation species world wide (Sabo and Knezevic, 2019).

E.camaldulensis plant is used to manufacture medications for a number of illnesses, including those affecting the respiratory system (such as colds, coughs, and asthma), the digestive tract (such as colic, diarrhoea and dysentery), the throat, and the muscles and joints. The various secondary metabolites that are present in medicinal plants include tannins, terpenoids, alkaloids, flavonoids, phenols, and quinones. These metabolites, along with others like them, have been utilized for a very long time in traditional medicine to treat a wide range of infectious diseases and other conditions (Abdulrasheed et al., 2019).

Methanolic extracts of leaves of Eucalyptus camaldulensis was studied for in vitro microbial activities by agar dilution method. The phytochemical analysis of the crude extracts of the medicinal plants revealed the presence of saponin, saponin glycosides, steroid, cardiac glycoside, tannins, volatile oils, phenols and balsam (gum). The methanolic extracts of the two plants inhibited the growth of Bacillus subtilis and Staphylococcus aureus (Babayi et al., 2004).

Methanolic extract of eucalyptus shows antibacterial property almost all the pathogenic strains got effected. B.cereus shows inhibitory effect by showing zone of inhibiton of 16.17mm size and this control is more than the control by tetracyclin which is 15mm, likewise E.coli showed 13.22mm and S.aureus showed 11.25mm (Devi et al., 2019).

The primary goal of this research is to determine how well Eucalyptus camaldulensis inhibits the growth of pathogenic bacterial strains responsible for the world's worst infectious diseases. In the fight against deadly infections, this research would unquestionably help humanity.

2. Material and Methods

The microbiology lab in the zoology department at University of Sargodha was the setting for all of the studies detailed here

2.1. Bacterial strains sampling

Bacillus subtilis samples were taken out of the ground. The soil shake technique was used to isolate B. subtilis on nutrient agar. (Jamil et al., 2007). From a rice field, Bacillus cereus was isolated phosphate buffer were combined with the calculated sample and serial dilution was carried out. Fresh milk and newborn powdered milk samples were separated to get a sample of Staphylococcus aureus. From fecal-contaminated water, Escherichia coli was isolated from feces-contaminated water. The sample was left at room temperature for a whole day. To reduce the amount of bacterial strain, serial dilutions of all the samples were carried out.

2.2. Collection of plant material

Fresh leaves of eucalyptus were collected from the University of Sargodha Punjab-Pakistan. Fresh leaves were picked and sun dried for 15 days and was grinded to get fine powder. 80 gm of the dried leaves were used for the extraction. The extraction from the leaves was done with the help of soxhlet apparatus (AL-Chaabawi and Satmbekova, 2023).

2.3. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) assay

Different concentrations 150 mg/mL, 180 mg/mL, 200 mg/mL, 220 mg/mL, and 250 mg/mL were used against bacterial strains. It exhibit growth inhibitory effects in all doses on nutrient agar plates. For MIC and MBC gar well diffusion assay was employed. Deionised water was used as negative control. After that, culture medium was maintained for 24hours at 37 °C. (Khusro et al., 2013).

2.4. Effect of temperature and pH

To test the impact of temperature, methanolic extract was subjected to five different temperatures (60 °C, 80 °C, 100 °C, and 121 °C). Water bath was used to maintain the temperature. (El-Mahmood, 2010). To study the extract effect at variable pH levels, five different pH levels were checked 3, 5, 7, 9, and 111. By adding sodium hydroxide and sulfuric acid, the pH of the solution was adjusted (Doughari et al., 2008).

2.5. Phytochemical screening

Phytochemical screening of eucalyptus extract was done by using qualitative methods. The techniques used to detect the presence of different phytochemical compounds are described below:

2.5.1. Qualitative methods

The phytochemical tests were performed by following Harborne (1973).

2.5.1.1. Saponins test

2 mL of extract was dissolved in 3 mL distilled water and shaken robustly. If saponins are presence of saponins was observed as a stable top layer of foam.

2.5.1.2. Hansch test for carbohydrates

2 mL of extract was taken in a test tube. 1 mL of concentrated H₂SO₄ was added from the side walls of the test tube and the formation of a brown ring suggested the presence of carbohydrates.

2.5.1.3. Tannins test

0.5 mL of extract solution was mixed with 1mL of water and 1-2 drops of FeCl₂ solution was added. Gallic tannins appeared blue while and catecholic tannins in green black colour.

2.5.1.4. Flavonoid test

2 mL of extract filtrate was taken. Then added 5-6 six drops of concentrated HCl and a few magnesium filings to observe the color changes. Presence of flavonoids was observed by the appearance of red color.

2.5.1.5. Phenol test

2 mL of plant extract was mixed with a pinch of ferric chloride. Presence of phenols was observed by the appearance of green color.

2.5.1.6. Protein test

2 mL of plant extract was taken, and mixed with1-2 drops of nitric acid. Development of yellow color indicates the incidence of proteins.

2.5.1.7. Quinone test

2 mL of plant extract was taken, few drops of concentrated H₂SO₄ were added. Appearance of red color indicates the presence of quinones.

2.5.1.8. Fat test

The plant extract was placed on the filter paper. Appearance of oil on the filter paper showed the presence of fat.

2.6. Statistical analysis

One way ANOVA, followed by the Tukey's test, was used to compare the means of the zones of inhibition of methanolic extract of clove and eucalyptus at various concentrations, temperatures, and pH levels.

3. Results

Using methanolic extract on an Escherichia coli plate, It was discovered an inhibitory zone with a minimum size of 4.1 mm at the concentration of 200 mg/mL and a maximum zone of 5.6 mm at 250 mg/mL, variable trend was found with increase in the concentration of extract. Similarly, Bacillus cereus has maximum sized zone of inhibition 3.6mm at the concentration of 200mg/mL and 1.3 mm wide at low concentration of 150mg/mL it shows the variable trend with increase in concentration. Bacillus subtilis has maximum 7.2mm-sized zone of inhibition at 180mg/mL and a minimum size 3.6mm zone of inhibition at 220mg/mL so variable trend was found. At minimal concentration, Staphylococcus aureus exhibits a 1.2 mm-wide zone of inhibition at minimum concentration of 150 mg/mL, whereas at highest concentration 25mg/mL, it measures 3.1 mm it shows the increasing trend with increase in concentration (Figure 1).

Figure 1
Inhibitory Zones by selected bacteria with different concentrations of Methanolic extract of E. camadulensis.

Zone inhibition measures for Bacillus cereus at the maximum zone of 6.4 mm was observed at 80 °C. Bacillus subtillis had a zone of inhibition of 4.6mm at 40 °C at the highest concentration of 250 mg/mL. E. coli showed zones of 5.4mm at 40 °C, 6.1mm at 60 °C, 6.2mm at 80 °C, 5.7mm at 100 °C, and 4.5mm at 121 °C. For Staphylococcus aureus, the maximum zone of inhibition was reported to be 6.6mm at 80 °C. The findings suggest variable trend of bacterial inhibition at different temperature of plant extract. The extract was observed to be optimum effective at 80 °C at 150 mg/mL concentration and at 40C at 250 mg/mL (Figures 2 and 3).

Figure 2
Methanolic extract of E. camadulensis at varying temperature with 150 mg/mL concentration.

Figure 3
Methanolic extract of E. camadulensis at varying temperature with 250 mg/mL concentration.

Inhibitory zones for Bacillus cereus were measured to be 4.1 mm at 3 pH, 5.2 mm at 5 pH, 6.2 mm at 7 pH was the lowest growth, and 4.3 mm at 9 pH, It measures 1.8mm and has a pH of 11 showing the maximum growth of strains, variable results were found. At a pH of 3, the inhibitory zone for Bacillus subtilis was 4.1 mm at 5 pH, 7.2 mm at 7 pH, 6.5 mm at 9 pH, 4.1 mm at 9 pH and 1.6mm wide at pH 11 which was minimum zone of inhibition, variable results were found. In E.coli 5.4mm at 3 pH, 4.7mm at 5 pH, 2.8mm at 7 pH, 3.7mmat 9 pH, and 9 pH and 2.0mm at 11 pH the lowest sized zone of inhibition was observed .In the same way Staphylococcus auresus 5.1 mm at 3 pH,, 5.3 mm at 5 pH, 8.1 mm at 7 pH, and 4.6 mm at 9 pH, and 1.5 mm at 11 pH restricted zone of inhibition was found respectively. At the basic pH of 11 growth was maximum its mean basic media is least effective in restraining the growth of all strains (Figures 4 and 5). Results of phytochemical screening are shown in Table 1. It was noted that eucalyptus methanolic extract have no traces of proteins while all other phytochemical constituents were found present in the plant extract.

Figure 4
Methanolic extract of E. camadulensis at varying pH with 150 mg/mL concentration.

Figure 5
Methanolic extract of E. camadulensis at varying temperature with 250 mg/mL concentration.

Table 1
Phytochemical screening of eucalyptus.

S.No	Phytochemical constituents	Methanolic extract of Eucalyptus
1	Quinones	+
2	Saponins	+
3	Hansch carbohydrates	+
4	Tannins	+
5	Phenols	+
6	Flavanoids	+
7	Proteins	-
8	Fat	+

• Present (+) Absent(-).

4. Discussion

E. camaldulensis was used in this investigation. The strains Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Bacillus cereus were used. Distinct zones of inhibition for each of the four different types of bacteria were observed by using methanolic extract of E.camaldulensis. In terms of concentration and efficacy. These extracts have proved their antibacterial potential.

Chuku et al. (2016) reported MICs ranging from 0.04mm to 200 mg/mL against B.subtilis and 1.25mm to 25 mg/mL against S.aureus, respectively by using methanolic E.camaldulensis extract. This study exhibits zone of inhibition against all the four strains including B.subtilis 7.2mm size MIC at 180 mg/mL and S.aureus 3.1mm size MIC as well in between the concentration range from 150 mg/mL to 250 mg/mL.

E.camaldulensis has been shown to be effective against both Gram-positive and Gram-negative bacteria within range ranged from 10 to 200 mg/mL. The efficacy varies greatly depending on how the extract was made. In the same vein, this research finds that the methanolic extract of eucalyptus is effective against both Gram-positive and Gram-negative strains of bacteria when the concentration is in the range of 150 mg/mL to 250 mg/mL (Abubakar, 2010).

This investigation lends support to the conclusions of Sabo and Knezevic (2019) as well. Sabo and Knezevic (2019) determined (MIC) for Bacillus subtilis was 5.0 mm. In this research Bacillus subtilis was treated with a concentration of 200 mg/mL in order to generate an inhibitory zone of 7.0 mm

Cimanga et al. (2002) provided evidence that an essential oil derived from E. camaldulensis have antimicrobial properties against E. coli as well as S. aureus with a zone diameter of inhibition ranging between 10 -12 mm and 18-30 mm, respectively. The results of this latest research indicate that the findings of Cimanga are valid because when the methanolic extract was employed the growth of E. coli and S. aureus was inhibited ranging 5.2-7.1mm and 1.2- 6.2mm.

Plant extracts from the Eucalyptus camaldulensis tree were examined by Reda et al. (2017) against several bacteria. Screening for Gram-positive bacteria often includes S.aureus. Most investigations found relatively strong activity against this bacteria, with minimum inhibitory concentrations in the 0.07–0.5% range. Confirmation of antibacterial action was also obtained against B.subtilis (0.17-0.34%), M.luteus (0.04%), and S. pyogenes (0.4-1.1%), in addition to S.aureus. In this study, a MIC of 0.2% was most effective for Bacillus cereus to produce a zone of inhibition of 3.6mm size. A MIC of 0.18% was proven effective in producing the zone of inhibition of 7.2mm in the case of Bacillus subtilis. A MIC of 0.25% was required to produce a zone of inhibition measuring 5.6mm in size in Escherichia coli. A MIC of 0.25% was required to produce a zone of inhibition measuring 3.1mm.

The presence of phytochemical components in E. camaldulensis leaf extracts is likely responsible for the antibacterial activity of these extracts. Pandey and Singh (2014) investigated the effectiveness of crude extract. Zone of inhibition of 1.5cm was produced against B. subtilis. According to the findings of this research, the growth of Bacillus subtilis was limited to a maximum zone of inhibition of 7.2mm and a minimum zone of inhibition of 3.6mm. Additionally, gram positive and negative bacteria were almost similarly suppressed.

Experiments on concentration that Babayi et al. (2004) carried out using eucalyptus methanolic extract revealed notable outcomes for all four bacterial strains that were examined in this work. The least concentration that was effective against Bacillus subtilis was 170 mg/mL, which resulted in an inhibitory zone that was 7.2 mm in size. The zone of inhibition against Bacillus cereus was 1.6 mm, while the zones of inhibition against E. coli and S. aureus were 4.5 mm and 1.2 mm, respectively. In this investigation, the lowest effective concentration that was employed was 150gm/mL, which created an inhibitory zone of 1.3mm against Bacillus cereus, 1.2mm against Staphylococcus aureus, 5.2mm against E. coli, and 6.2mm against Bacillus subtilis.

According to the findings of previous studies, the antibacterial activity of the methanolic extract. This control is greater than the control provided by tetracyclin, which is 15mm; similarly, E. coli showed 13.22mm and S. aureus showed 11.25mm. B. cereus demonstrated an inhibitory effect by exhibiting a zone of inhibition measuring 16.17mm in size; this control is superior to the control provided by tetracyclin, which is 15mm. This study also demonstrates the impact of methanolic extract on E. coli with a measurement of 5.2 mm, Staphylococcus aureus with a measurement of 1.2 mm, Bacillus cereus with a measurement of 1.2 mm, and Bacillus subtilis with a measurement of 6.2 mm.

E. camaldulensis crude leaf extracts were tested for antibacterial efficacy at pH 2 and 10 studied by El-Mahmood (2010) Zones of inhibition of 7 mm for E. coli at pH 2 and 12 mm at pH 10; 8 mm for P. mirabilis at pH 2 and 14 mm at pH 10; 10 mm for S. typhi at pH 2 and 14 mm at pH 10; 11 mm for S. aureus at pH 2 and 13 mm at pH 10, and 9 mm for K. pneumoniae at pH 2 and 15 mm at pH 10. Under acidic circumstances, all of the extracts were less active than they were in alkaline ones. But in this research results are variable in the case of Bacillus cereus by employing the 150 mg/mL concentration increase in pH resulted in decrease in zone size but in case of Staphylococcus aureus 9 pH given the maximum zone size of 6.1mm. Neutral pH showed the maximum zone size in Bacillus subtilis.

Temperature's impact on the extracts' potency was studied by Doughari et al. (2008). Zones of inhibition with diameters of 9 mm at 100 °C for E. coli and 14 mm at 100 °C for P. mirabilis, 11 mm at 100 °C for S. typhi, 10 mm at 100 °C for S. typhi, 17 mm at 100 °C for S. aureus, and 14 mm at 100 °C for K. pneumoniae. The effectiveness of the antibacterial agent decreased with increasing temperature. In this research in the case of E.coli with the increase in temperature zone size decreased from 40 °C to 121 °C by using concentration of 150 mg/mL. Other three bacteria B.cereus, B. subtilis and Staph.aureus shows the variable trends.

5. Conclusion

The effectiveness of eucalyptus plant as antibacterial agent is evaluated here. This research reveals that eucalyptus methanolic extract is very efficient at inhibiting the development of several bacteria, including but not limited to E. coli, Bacillus cereus, Bacillus subtilis, and Staphylococcus aureus. It demonstrates that secondary metabolites in this plant are useful antibiotics that would be useful in making new drugs for humans and animals.

Acknowledgements

We want to acknowledge Department of Zoology, University of Sargodha, Sargodha, Pakistan for providing all facilities to perform this research work.

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Publication Dates

• Publication in this collection
  22 Nov 2024
• Date of issue
  2024

History

• Received
  31 Jan 2024
• Accepted
  12 Aug 2024