Citrus EOs are one of the main by-products of the orange juice industry and could have several applications besides cosmetology and the food industry. Citrus oils have been reported as having antimicrobial properties20,21,22,25, but testing their antimicrobial activity on bacteria affecting animal farms has not been greatly mentioned. E. coli causing post-weaning diarrhea in piglets during their early life is a critical problem for the pig industry2. The antibacterial activity of commercial citrus EOs evaluated in this study on E. coli strains isolated from pig gut and on Lactobacillus bacteria showed that, in overall, citrus EOs presented a selective antibacterial activity, having higher activity on pathogenic bacteria E. coli than on beneficial bacteria Lactobacillus spp. The selectivity towards bacterial pathogens by EOs or EO compounds rather than beneficial bacteria have been already reported18,26,27, EOs or single EO compounds were shown to have higher inhibitory effect on E. coli O157:H7, E. coli K88, S. typhimurium than on Lactobacillus and Bifidobacterium spp18. As an alternative to AGPs, the selectivity of citrus oils could be an important feature of antimicrobial spectrum, which commonly has not been considered to conventional AGPs long used, since the aim is to have an effect on the pig gut. Thus, current search for antimicrobial substances as alternatives to AGPs should consider selectivity aspects between pathogenic and beneficial gut bacteria. The lower effect on beneficial bacteria by an AGP and its potential alternatives (such as citrus EOs) would be desirable since beneficial bacteria such as Lactobacillus spp. contribute to fighting pathogen colonization in the gut and preventing gut infections. Thereby, reinforcing gut microbiota and contribute to improve animal health27.
The capability of citrus EOs to inhibit pathogenic bacteria has been well reported in several studies. For instance, tangerine EO (Citrus reticulata) was reported as having an inhibitory effect on S. aureus, Bacillus subtilis, and Pseudomonas aeruginosa28. Another study proved the high effectiveness of the lemon EO (Citrus limon L. Burm) to inhibit several strains of Listeria monocytogenes, S aureus and Salmonella enterica associated with foodborne diseases25 Also, it has been reaffirmed the anti-salmonella22 and anti-listeria21 activities of several commercial citrus oils. Specifically, the antibacterial activity of citrus oils on E. coli has been investigated. Tangerine EO (Citrus reticulata) was shown to be effective to produce an inhibition of 14.6 ± 1.1 mm on E. coli28. A total inhibition of E. coli growth by this oil was found at 1.96 mg/mL29. A close value to this MIC was found for BOT oil in our study (1.85 mg/mL = 0.21%v/v). Other citrus EOs such as bergamot, orange and lemon were also effective to produce inhibitions ≥18 mm on E. coli O157, but full inhibition of the growth of this bacterium was reported at higher MICs than the BOT oil, between 0.5–1.0%v/v30. Similarly, another study reported higher MICs than the BOT to mandarin and lemon EOs on E. coli, 5 and 30 µL/mL, respectively31. In addition, the EO of sweet orange (Citrus sinensis Osbeck) has presented an inhibitory effect on E. coli at 18.8 µL/mL32, also considered as a high MIC in contrast to the BOT oil. Conversely, the non-effectiveness of several citrus oils to fight E. coli affecting animals, such as E. coli associated with poultry colibacillosis, has been reported33. In comparison, the citrus EOs tested in our study were quite effective in treating E. coli affecting pigs.
Furthermore, some studies highlighted that citrus EOs have higher effectiveness to inhibit Gram-positive pathogenic bacteria than Gram-negative pathogenic bacteria30,34. However, in our study, the opposite was observed, since the Gram-negative E. coli was more sensitive than the Gram-positive Lactobacillus spp. to the activity of the citrus oils. The difference in the sensitivity to EOs between these two groups of bacteria has been hypothesized to be the consequence of differences in the cell wall structure, since Gram-positive bacteria lack an outer membrane (OM), which Gram-negative bacteria have. This OM contains lipopolysaccharides (LPS) with polar ends (O-polysaccharides) and transmembrane channels (porins), which permit the passage of hydrophilic solutes and make difficult for hydrophobic compounds to diffuse such as EOs components into the cell. Therefore, this would allow Gram-negative bacteria be more resistant to EOs35. Nonetheless, the antibacterial spectrum of EOs depends on the specificity of the functional groups of EO compounds to single or multiple targets. Some EO compounds have the ability to disintegrate the OM of Gram-negatives as E. coli, release the material associated to this membrane and penetrate the cell, provoking a disruptive effect36. Probably, the compounds present in citrus EOs may have this ability due to their higher effectiveness observed on this Gram-negative bacterium, E. coli. On the other hand, the antibacterial activity of citrus oils on Gram-positive beneficial bacteria has been little reported. Orange, lemon, mandarin and grapefruit EOs had a low inhibitory effect on Lactobacillus sakei and Lactobacillus curvatus, exhibiting the orange oil the lowest effect on these bacteria (12.8 ± 0.5 and 13.5 ± 0.2 mm, respectively)37. The authors demonstrated that the inhibitory effect of these four oils was dose-dependent causing inhibition of those Lactobacillus species only at the highest concentrations tested37. This was also noticed in our study, where the citrus oils exhibited IZDs lower than 11 mm to Lactobacillus species. Moreover, L. rhamnosus was inhibited at a high BOT concentration and killed even at an upper concentration, thus showing BOT had a low antibacterial activity on L. rhamnosus. Although general structures and biosynthesis pathways among Gram-positive bacteria are conserved, some Gram-positive bacteria, such as Lactobacillus spp., could show low sensitivity to antimicrobials, such as EOs, since the cell wall of Gram-positive lactic acid bacteria (LAB) as Lactobacillus spp. possess unique properties that could confer intrinsic resistance to some antimicrobial agents38. For instance, the intrinsic resistance to antibiotics of some Lactobacillus (e.g. to vancomycin) would be related to the fact of having a D-lactate instead of D-alanine as the last amino acid in the peptidic chain of the peptidoglycan layer of their cell wall38,39, which would avoid the antibiotic binds to the peptidic chain and cause the inhibition of these bacteria40.
Additionally, it was observed that BOT oil caused higher disturbances on the growth kinetics of E. coli than L. rhamnosus, significantly affecting its maximal culture density and the lag phase duration. Both parameters were dose-affected and changed as function of the BOT concentration. The higher dose-dependent effect of some EOs and single EO compounds on the growth kinetic of E. coli than on Lactobacillus spp. has already been observed27. Oregano, thyme and rosemary EOs, carvacrol, eugenol and thymol provoked higher reduction on the maximal culture density of E. coli strains than Lactobacillus fermentum and Lactobacillus reuteri with increasing of the concentration of EOs/EO compounds27. Also, the dose-dependent effect of carvacrol to extend the lag phase of E. coli by increasing the concentration of this compound has been proved41. In addition, some combinations of EOs have been reported as more efficient to cause an increase of the E. coli lag phase. For instance, combinations of oregano with basil EOs and oregano with lemon balm EOs were able to significantly increase the E. coli lag phase, approximately 7.4 h and 3.6 h longer, respectively, compared to when oregano EO was used alone42. Regarding L. rhamnosus, in our study, we observed only the lag phase duration was extended as the BOT concentration was increased. This effect on L. rhamnosus has been previously observed with the oil of Melaleuca armillaris, which additionally reduced the growth rate and final culture density with increasing of the EO concentration43. Therefore, this oil had a higher dose-dependent effect on the growth kinetic parameters of L. rhamnosus than the citrus oil (BOT) tested in our study. Moreover, a recent study observed that Eucalyptus globulus and Pimenta pseudocaryophyllus EOs presented a dose-dependent effect on the lag phase of L. rhamnosus as well; however, P. pseudocaryophyllus oil caused higher extension of this parameter in comparison to E. globulus oil at the same sub-MICs44.
Limonene has been shown as a major compound of citrus EO composition and most of their biological activities have been attributed to this compound. All citrus oils evaluated in our study presented limonene as the major compound. However, the mismatching between limonene content and antibacterial activity of these oils suggested that their antibacterial activity cannot be attributed exclusively to limonene. Some studies have already reported the lack of antibacterial activity of limonene individually tested. For instance, the pompia EO (Citrus limon var. pompia), which presented limonene as major compound (28%), at a concentration of 256.3 mg/mL, presented an antibacterial effect on several pathogenic bacteria, but when limonene was evaluated alone, it did not exhibit any antibacterial effect45. Thus, this proved that limonene would not be the compound responsible for the antibacterial activity observed for this oil. Nonetheless, coexisting minor compounds in citrus oils could contribute to conferring the antibacterial property of these oils. In mandarin EO, compounds like octanal, decanal, citral, citronellal, linalool, α-sinensal and thymol were suggested as possible collaborators to the antibacterial activity, when this oil (with 56.8% of limonene) was tested against Gram-negative and Gram-positive bacteria28. Other minor compounds, belonging to oxygenated monoterpenes class, such as 4-terpineol, α-terpineol, cis-geraniol, β-citral, nerol and α-citral, might also be implicated in conferring the antibacterial activity of citrus oils, since they have been detected in high amounts in the composition of the citrus EO that presented high antibacterial activity25. Minor oxygenated monoterpenes compounds (cis-limonene oxide, trans-carveol, carvone, trans-limonene oxide and perrilla alcohol) were also detected in the group of the most selective citrus oils of our study. Possibly, these compounds might play an important role in conferring the selective antibacterial activity of citrus EOs. In addition, an orange cold pressed EO rich in limonene (85.3%) presented an antibacterial activity on E. coli ten times higher than limonene alone, and even minor compounds, such as linalool, pinene and terpineol, presented a higher activity than limonene46. Furthermore, the antibacterial activity of limonene has been shown to be variable and depending on its stereoisomeric form present in the EO. The (−) stereoisomer of limonene could inhibit E. coli at a lower concentration (8 mg/mL) than the (+) stereoisomer (11 mg/mL). On the other hand, limonene alone has been proved to stimulate the growth of beneficial bacteria as L. fermentum, instead of having any inhibitory effect on it27. Likewise, limonene has been reported as not effective to inhibit several Lactobacillus species including L. rhamnosus ATCC 746945, the bacteria also evaluated in our study, and which showed be the more resistant to the antibacterial activity of the citrus oils proved. Therefore, it would be possible to infer that limonene could collaborate with the selective activity of citrus oils when it is present in the gut, promoting the beneficial bacteria while other minor compounds could act in inhibiting pathogenic bacteria. It has been reported that in an EO, major and minor components probably act in synergism to confer the biological properties of the EO47. When an EO compound is proved, individually, its effect may differ from the effect that this compound may have in combination with the other compounds inside the EO. Thus, it would be recommended the use of the whole EO instead of single EO compounds, since every compound inside an EO could exert a different mechanism of action on the bacteria cell48, and this could reduce the chance to bacteria develop easily resistance to the EO. Contrariwise, bacteria could develop a rapid and easy mechanism of resistance to a single EO compound, as in the case of an antibiotic, which consist of a single compound. Several mechanisms of action of EOs have been proposed in the literature. The mechanism of action comprises a serie of events on the bacterial cells. Initially, they can destabilize the cellular architecture, leading to the breakdown of membrane integrity and thus increased permeability of cellular constituents. This disrupts many cellular activities, including energy production, membrane transport, and other metabolic regulatory functions48. In addition, EOs can alter the membrane fatty acids composition, the membrane proton motive force and affect proteins in the cytoplasmatic membrane. Additionally, EOs can interfere with the quorum sensing activity, decreasing the proteolytic activity, biofilm formation, and virulence factors expression and their functions, as well as to affect the metabolome of bacteria35.
In conclusion, our study highlights as an important feature of antimicrobial spectrum the selectivity between pathogenic and beneficial gut bacteria, which should be considered when searching for antimicrobial substances as alternatives to conventional AGPs, since the aim is to have an effect on the pig gut. Our study has proved, by a screening, MIC determination, and growth kinetic parameters evaluation, the selective antibacterial activity of citrus EOs on E. coli and Lactobacillus spp., thus suggesting these EOs as potential alternatives to AGPs. Consequently, based on the selective performance and the huge viability in the global market of citrus EOs, the possible application of these oils in the pig production sector could turn feasible. In addition, chemical composition characterization showed that minor compounds present in these citrus oils would be implicated in conferring their selective activity, instead of limonene, the major present compound, playing this role exclusively. Finally, our results motivate further research to clarify, for instance, the possible mechanism of action that citrus oils would have on pathogenic and beneficial bacteria as well as their direct effect on the pig gut and on the microbiota resident in it.