1 Introduction

The burden of infectious diseases is on the rise globally, and this burden is worse in the tropics, where bacterial infections play a key role [1]. In 2019, bacterial infections were responsible for one out of every eight deaths in humans. Furthermore, deaths due to bacterial infections were the second leading cause of global deaths in the same year [1]. A recent global analysis of 33 bacterial pathogens revealed that the five leading bacterial agents causing death in humans were Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Klebsiella pneumoniae and Pseudomonas aeruginosa [1]. These five pathogens account for more than 50% of the global bacteria-related deaths, with the mortality rate highest in sub-Saharan Africa and lowest in high-income countries [1]. Similarly, an assessment of the health burden of bacterial infections in Europe from 2016–2020 not only indicated an increasing burden on infants and elderly individuals but also revealed an increasing number of infections resistant to very costly antibiotics [2].

Although a WHO 2021 report indicated a decrease in the mortality rate caused by infectious diseases, nevertheless, the report indicated that infectious diseases still accounted for 6.2–16.7 million deaths in 2019, which represented 18% of all deaths. This is an indication that these diseases are still very common among humans [3]. Furthermore, a recent report indicated that diarhoea and other infectious diseases, including neonatal disorders, accounted for more than 60% of communicable disease disability-adjusted life years (DALY) losses globally in 2021[4]. These observations attest to the enormous burden of infectious diseases worldwide and the need for concerted efforts to stem their spread.

Plants are potential sources of promising antimicrobial agents. Citrus plants are a class of plants belonging to the Rutaceae family and include approximately 1300 species. One of the important products of citrus fruits is essential oil, which is obtained mostly from the peels or seeds of these fruits, and these peels and seeds are often considered waste products [5]. Citrus essential oils are a mixture of volatile compounds that are composed mainly of monoterpenes and various other compounds that are potential sources for screening anticancer, antimicrobial, antioxidant and free radical scavenging agents [6, 7]. Essential oils are also rich in flavonoids, terpenes, carotenes and coumarins, which have a variety of biological effects and are responsible for antimicrobial activities, revealing their potential for therapeutic applications [8, 9].

The increase in the emergence of new bacterial strains that are multi resistant coupled with the nonavailability and high cost of new generation antibiotics has resulted in increased morbidity and mortality [1, 2]. The high cost of antibacterial agents makes them elusive and unsustainable for the care of patients in resource-poor settings in most sub-Saharan countries. Therefore, there is a need to identify inexpensive, effective, and sustainable means of combating these bacterial diseases in the poor setting in most developing countries. This is more important for sub-Saharan Africa, where the burden of infectious diseases is quite high and there are limited tools and resources available to combat this disease [1, 10].

Despite the great potential of plant products as antibacterial agents for the treatment of common human infectious diseases, available data point to their gross underutilization as only 3% of the antibacterial agents approved by the Food and Drug Administration (FDA) in the U.S.A. are from plant natural products [11, 12]. Therefore, exploring the potential of locally available plant products otherwise considered waste products in resource-poor settings in sub-Saharan Africa may be a good step in exploring the potential of some neglected plant products for tackling the global burden of infectious diseases in these resource-poor settings.

The aim of this research was to investigate the activity of essential oils from several citrus species against six common pathogenic bacteria in the tropics. The six pathogenic bacteria were purposively selected because they are very common worldwide, especially in the tropics, where they contribute immensely to the high burden of bacterial infections.

2 Materials and methods

2.1 Sample collection and identification

The citrus species used in this study were lime (Citrus aurantifolia), grapefruit (C. paradisi) and tangerine (C. reticulata). These samples were collected from various farms in the Ado Ekiti metropolis and were identified at the Department of Plant Science of Ekiti State University, Ado Ekiti, Ekiti State, Nigeria, with herbarium numbers UHAE 2023035, UHAE 2023036, and UHAE 2023037. The fruits were washed several times under running water. The peels were subsequently removed and divided into two parts. One part was ground immediately with an electric grinder, and the essential oil was extracted, while the other part was air-dried before extraction of the essential oil.

2.2 Extraction of essential oil

The essential oil was extracted using Clevenger—type apparatus for 3 h as described by the European Pharmacopoeia [13]. After extraction, the essential oil was collected and dried over anhydrous sodium sulfate. The solution was stored at − 4 °C until further use.

2.3 Determination of antimicrobial activity of the essential oils

The microorganisms used for this investigation were Staphylococcus aureus (ATCC 25923), Streptococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Bacillus subtilis (ATCC 19659-0540P), Escherichia coli (ATCC 25922) and Klebsiella pneumoniae (NCTC 7427), which were isolated from clinical specimens. Clinical isolates of the bacteria were obtained from the microbiology and parasitology laboratory of The Wesley Guild Hospital, Ilesa, a unit of The Obafemi Awolowo University Teaching Hospital Complex Ile Ife Osun State, Nigeria. The modified agar well diffusion method of Murray et al. [14] was used to determine the antimicrobial activity of the oils. Twenty milliliters of nutrient agar was dispensed into sterile universal bottles, inoculated with 0.2 ml of bacterial culture mixture gently and poured into sterile Petri dishes. A number three-cup borer (6 mm diameter) was properly sterilized by flaming and used to make three to five uniform cups/wells in each petri dish which was sealed with a drop of molten nutrient agar, and the cups/well were filled with 50 μl of the extract and allowed to diffuse for 45 min. The Petri dish was incubated at 37 °C for 24 h for each bacterial colony, and the same procedure was carried out for the reference antibiotics. Antimicrobial activity of the EOs was evaluated by measuring the diameter of the growth inhibition zones in millimeters. The experiment was repeated three times. The reference antibiotics used were tetracycline, streptomycin, amoxicillin/clavulanic acid, azithromycin, cefuroxime, ciprofloxacin, ceftriaxone, ampicillin/cloxacillin (Ampicillin), metronidazole, amoxicillin, trimethoprim-sulfamethoxazole, and gentamicin. The antibiotics used were purchased from the pharmacy unit of the Ekiti State University Teaching Hospital, Ado Ekiti and are commonly used to treat common infectious diseases in Nigeria.

The sensitivity of each bacterium to the essential oils was ranked based on the inhibition zone values expressed in millimeters (mm) according to Clinical and Laboratory Standard Institute [15] as follows: susceptible for zones with a diameter ≥ 20 mm, intermediate for zones with a diameter between 15 and 19 mm, and resistant for zones with a diameter ≤ 14 mm.

2.4 Assessment of the minimum inhibitory and bactericidal concentrations

The broth microdilution method was used to determine both the MIC and MBC as described by Bouhdid et al. [16], with slight modifications. A stock solution of each extract was prepared.

A volume of 50 µl per well was prepared from serial dilutions of the stock solution to give concentration ranging from 2 to 0.0010% v/v which was prepared in sterile 96—well plates. Then, 50 µl of LB broth inoculated with the bacterial suspension adjusted to 106 CFU/mL was added to each well and incubated at 37 °C for 18 h. The bacterial growth of each well was rated by the addition of 5 µl resazurin and then the mixture was incubated for another 2 h. The MIC was taken as the lowest concentration of essential oil that did not show any change in resazurin color. Approximately 10 µl from negative wells was sub-cultured on plate count agar (PCA) medium and incubated for 24 h to carry out the broth dilution tests. The MBC was taken as the lowest concentration of EO with no visible bacterial colonies after 24 h of incubation.

2.5 Statistical analysis

Analysis of variance was performed using GraphPad Prism Version 5.0 Statistical Software for Windows (San Diego, California, USA) to determine the differences between the activity of the essential oils and that of the standard antibiotic agents on the basis of the microbial community. A p value was considered to indicate statistical significance at p < 0.05 with a 95% confidence interval.

3 Results

3.1 Physical properties of essential oils

The essential oils had a pleasant intense smell and were liquid at room temperature (25 °C). The oils maintained their state even after storage at − 4 °C.

3.2 Antimicrobial activity

The in vitro activity of the essential oils from the citrus groups against the six pathogenic bacteria showed that the essential oils had an average high zone of inhibition against the investigated bacteria (Table 1).

Table 1 Zone of inhibition of the essential oil (mm) against the tested microorganisms
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Fresh tangerine essential oil had the highest zone of inhibition against Staphylococcus aureus, followed closely by fresh and dried lime, while dried tangerine had the lowest zone of inhibition. Staphylococcus aureus showed susceptibility to essential oils, whereas for S. faecalis, the sensitivity ranged between intermediate and susceptible, with dried grapefruit EO having the highest zone of inhibition and fresh lime having the lowest. Overall, the sensitivities of the investigated bacteria to the essential oils extracted from the three citrus species ranged from susceptible to intermediate, with the lowest sensitivity observed for E. coli against fresh grapefruit EO, which has a zone of inhibition of 15 mm (Table 1).

3.3 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the essential oils

The MIC, MBC and ratio of the MBC to the MIC of the essential oils against the six tested bacteria are presented in Table 2. The MIC ranged between 0.025 and 0.15 mg/ml against all the tested microorganisms. The MBC:MIC ratio was low for all the essential oils, with values ≤ 3 (Table 2).

Table 2 Minimum inhibitory concentration, minimum bactericidal concentration and the MBC/MIC ratio for the EOs against the tested microorganisms
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3.4 Comparison of the activity of essential oils and several conventional antibiotic agents against the tested microorganisms

Some of the essential oils had higher activity against some microorganisms than did the standard antibiotics, as shown in Table 3.

Table 3 Comparison of the zones of inhibition of the EOs (mm) against the tested microorganisms and the conventional antibiotics
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Lime essential oils (fresh and dried) had higher activity than did some of the standard antibiotics except streptomycin, amoxicillin/clavulanic acid, ciprofloxacin and ceftriaxone when tested against Staphylococcus aureus. Additionally, all the EOs investigated showed activity against Streptococcus faecalis, E. coli and Bacillus subtilis, whereas no activity was detected against some of these organisms from some standard antibiotics. Overall, the performance of the essential oils was comparable to that of conventional antibiotics and seemed better than that of certain bacterial agents (Table 3).

4 Discussion

This study investigated several citrus essential oils antimicrobial activities against six common pathogenic bacteria and compared them to commonly used antibiotics. The MIC values showed that the activities of the EOs fell within the high activity range, with MIC values ≤ 2 mg/ml. The MBC also showed high activity, with an MBC:MIC ratio ≤ 2 for most of the tested essential oils, which indicates bactericidal activity, while very few fall between 2 and 3, which indicates bacteriostatic activity (Table 2), as previously described [17, 18]. These observations support the findings of Oyelami et al. [19] on the antimicrobial activity of grapefruit seeds in the treatment of UTI and of Adegoke et al. [20] on the antimicrobial/anti-infective activity of lime juice, thus revealing the potential of these essential oils for combating some diseases caused by these microorganisms in resource-poor settings in sub-Saharan Africa.

Furthermore, all the investigated essential oils exhibited high activity against all the bacteria in this study, and all of these bacteria could cause severe infections in humans. This observation further buttresses the potential of these plant waste products as possible candidates for combating antimicrobial scourge and associated diseases in humans. For example, S. aureus is a major human pathogen that can cause a wide variety of infections [1], hospital-acquired infections, sepsis, toxic shock syndrome, skin infection, osteomyelitis and urinary tract infection (UTI), among others [1, 19, 21]. This bacterium has been found to be increasingly resistant to several commonly used and available antibiotics through several mechanisms [1, 21]. The observation that this microorganism is susceptible to all the investigated essential oils is noteworthy and shows that these essential oils could be potential sources of good antibiotics that could help to combat some of the diseases caused by S. aureus. The activities of these essential oils, especially dried lime, fresh tangerine and fresh lime, against this bacterium (S. aureus) may be connected to the components of these essential oils, as all of them have been found to possess a high percentage of monoterpene and oxygenated monoterpene hydrocarbons, which have been shown to have good antibacterial activity [5, 22, 23].

Notably, the essential oils have good activity against enterobacteria, E. coli and K. pneumoniae, both of which are causes of serious human infections and hospital-acquired diseases, which are the leading causes of morbidity and mortality in humans [1,2,3]. The activity of dried tangerine and dried grapefruit against E. coli was greater than that of some of the conventional antibiotics except streptomycin, ceftriaxone and ciprofloxacin (Table 3). Similarly, K. pneumoniae was susceptible to all the EOs, and this was also observed to be prominent in the activity of dried grapefruit against the bacterium, which was comparable to that of ciprofloxacin and clavunated amoxicillin ciprofloxacin but also higher than that of some of the standard antibiotics except streptomycin, ceftriaxone and gentamicin (Table 3). These observations highlight the potential of these essential oils for combating infections caused by the above organisms.

Given the recent evidence suggesting the increasing emergence of resistant strains of these organisms [1,2,3, 24], it is postulated that these infections could strongly limit health facilities in the developing world [1]. Furthermore, by virtue of their roles in nosocomial infections, these organisms also contribute to increasing the cost of care for patients through increasing their morbidities, staying in the hospital, and using expensive drugs. Therefore, exploring the potentials of EOs from these underutilized plant waste products could be a good way to combat these dangerous infectious agents.

To further demonstrate the great potential of the studied plant EOs against a wide range of organisms, they were tested against both P. aeruginosa and Streptococcus faecalis, both of which are susceptible to grapefruit EOs (both dry and fresh) and dried tangerine EOs. In addition, they had intermediate sensitivity to other investigated EOs (Table 3). Again, these observations demonstrated the great potential of oils from the plant waste materials examined in this study. Pseudomonas aeruginosa causes major ill health like nosocomial infections, sepsis, UTIs, and ear infections in children and adults [1, 24, 25]. Additionally, Streptococcus faecalis can cause severe infections in the liver and heart. Pseudomonas aeruginosa has a very restrictive outer membrane and also possesses other resistance mechanisms which make the organism resistance to many antibiotics [25]. Hence, the observed sensitivity of these EOs to P. aeruginosa is heartwarming and a reflection of the potential antimicrobial value of the EOs.

The observed activity of the EOs could be linked to the high amount of monoterpene hydrocarbons and other ethnobotanical compounds present in the essential oils, such as limonene, alpha and beta pinene, and alpha and beta terpineol, as shown in Fig. 1 [5, 22, 23]. In addition, the hydrophobicity and ability of EOs to easily diffuse into or penetrate through the damaged cell membranes of microorganisms, thereby inhibiting the activities of these microorganisms, could also be responsible for the observed activity of the essential oils [9, 26].

Fig. 1
figure 1

Structures of several compounds detected in the essential oils obtained from the investigated citrus species [5, 21]

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In general, our findings showed that there was no significant difference in the sensitivity of gram-negative and gram-positive bacteria to the essential oils, thus revealing that the EOs in the present study exhibited wide and broad-spectrum activity against all the tested microorganisms.

Notably, our findings in the current study are also in agreement with the reports of previous authors on the antibacterial activity of essential oils [23, 26,27,28,29,30].

Furthermore, and of great importance is the nonsignificant effect of drying on the efficacy of the essential oils (Table 1). As shown in this table, processing technique did not significantly impact the antimicrobial activities of the EOs, as the EOs exhibited high activity against the varieties of bacteria investigated. This observation is relevant in the developing world, where there are limited tools/infrastructures available for the various highly advanced technological procedures being performed in the developed world for industrial processing. Therefore, these findings could aid in the development of these materials for industrial application as candidate antibiotics in resource-poor settings in sub-Saharan Africa in the tropics because some of these samples are seasonal, and thus, storing the peels is unlikely to affect their antibacterial ability.

The relevance of the findings of the current study cannot be overlooked for several reasons. First, the plant products studied are considered waste products with little or no significant differences. Second, this study revealed that these otherwise neglected plant products could be potential sources of antimicrobial agents against some bacterial infectious diseases troubling humans. Third, the availability of these plant products can be guaranteed for sustained sources of antimicrobial agents if explored because the plants readily grow in sub-Saharan Africa. Last, it has been suggested that the rich composition of ethnobotanical constituents of plant products leaves little room for bacterial agents to develop resistance against them because the various constituents will exert antimicrobial effects through divergent targets, thus preventing the development of antimicrobial resistance against them [11, 12]. This could guarantee their continued usage for a long time, thus enhancing their potential for sustained relevance in combating the menace of infectious diseases in both developed and developing countries.

5 Conclusion

The essential oils from the three species in the citrus groups had remarkable antibacterial activity against the investigated bacterial agents. The antimicrobial activities of EOs from citrus species are comparable to those of conventional antibiotics; thus, EOs from citrus species have potential as materials of great, medicinal, industrial, and economic value for tackling the menace of some tropical bacterial infections.