Antibacterial Effect Of Natural Oils – An Opportunity To Solve The Problem Of Antibiotic Resistance On The Example Of Pseudomonas Spp.

P. aeruginosa, like most Gram-negative pathogens, shows an increasing resistance to the majority of standard antibiotics used. A particularly dangerous phenomenon is the accumulation of resistance after the exposure of these bacteria to antibiotics and cross-resistance between them. This is responsible for multi-drug resistance, inter alia, in P. aeruginosa. This situation is particularly worrying because it creates a state reminiscent of the pre-antibiotic era [8].

P. aeruginosa is a common cause of health care-associated infections, including pneumonia and infections of the bloodstream, urinary tract, and surgicalsite infections. Generally, more than 6,000 (13%) of the 51,000 health care-associated P. aeruginosa infections occurring in the United States each year are multi-drug resistant. Thus, infections caused by P. aeruginosa are responsible for 400 deaths per year [52]. In Europe, it is estimated that P. aeruginosa is responsible for 10% of all hospital-acquired infections, but this number is constantly on the rise [3, 8, 80]. The factors influencing the growing resistance in P. aeruginosa include the low permeability of its cell wall, the genetic capacity to express a wide repertoire of resistance mechanisms, the constitutive expression of various efflux pumps, and the production of antibiotic-inactivating enzymes. Moreover, the evidence that it can acquire additional resistance genes from other organisms via plasmids, transposons and bacteriophages is presented [48–49, 59]. Because of this complex multidrug resistance, P. aeruginosa is used as a model organism in research on resistance or susceptibility to synthetic or natural antibacterials [3, 5, 36, 47–49, 54, 81].

Microorganisms in every environment on earth are exposed to antimicrobials and therefore some species (such as P. aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia) possess an intrinsically low susceptibility to antibiotics. Free-living opportunistic pathogens often have large genomes that allow the colonization of diverse environments through metabolic versatility that helps to degrade and resist the toxicity of compounds present in these ecosystems. This can include large numbers of biodegradative enzymes which cooperate in the modification and utilization of antibiotics as a food resource. Additionally, efflux pumps – originally involved in signal trafficking or resistance to toxic compounds produced by plants or rhizosphere-associated microbiota – can be also used for effluxing antibiotics [41, 57, 77].

P. aeruginosa has been classified within the group of six highly antibiotic-resistant bacteria that are the primary causative agents of nosocomial (hospitalacquired) infections [66]. An understanding of how this intrinsic resistance works was not easy to come by and was tackled by Murray et al. [66]. Primarily, they assumed that this resistance is related to gene expression in the presence of nonlethal levels of an antimicrobial with the idea that genes which are differentially regulated by low levels of an antimicrobial will provide an immediate insight into factors important for intrinsic resistance. However, it turned out that most of the genes identified in these studies have proven not to be significant in terms of intrinsic resistance. Another idea of theirs was based on screening transposon mutant libraries for increased or decreased susceptibility to sub-MIC antimicrobials These studies revealed novel resistance determinants, although it is necessary to continue them in order to test more antibiotics and confirm all the results obtained [66].

The exposure of susceptible P. aeruginosa to an aminoglycoside results in rapid drug concentration-dependent killing followed by a phase of bacterial refractoriness characterized by slow drug concentration-independent killing. This adaptive resistance, which is distinct from the post-antibiotic effect and which disappears when the organism is no longer incubated with the aminoglycoside, has been observed in vitro, in animal models of infection, and in patients with cystic fibrosis. This adaptive resistance is certainly not a result of mutation events. Reduced intracellular accumulation of aminoglycosides (which is concomitant to adaptive resistance) was first interpreted as the consequence of a lower drug uptake across the bacterial envelopes. Supporting this assumption, pleiotropic changes in the protein profiles of the cytoplasmic membrane were detected in drug-exposed bacteria. The membrane potential – the driving force for drug entry – appears to be marginally diminished in adaptively resistant bacteria. This finding agrees well with the observation that surviving bacteria grow normally during the post-exposure refractory phase [37]. According to Lambert [49], aminoglycosides are not able to cross the outer membrane via porin channels. They promote their own uptake by binding to the lipopolysaccharide on the outer face of the membrane. Aminoglycosides destroys the permeability barrier of the outer membrane and allows the antibiotics to penetrate through the wall to the cytoplasmic membrane. Aminoglycosides are then actively transported into the cells, where they interfere with protein synthesis at the ribosomes. Lambert [49] stated that resistance to aminoglycosides in laboratory strains of P. aeruginosa can be observed due to the overexpression of an outer membrane protein, OprH, which protects the lipopolysaccharides from binding the antibiotics but he underlined that this form of resistance has not been encountered widely in clinical isolates.

Fig. 2.

In clinical practice, colistin and polymyxin B are most commonly used to treat P. aeruginosa infections. Thus, an increase in the number of polymyxins-resistant clinical isolates is being reported. The polymyxins are antimicrobial cyclic oligopeptides that interact with the phospholipids of bacterial cell membranes and thereby lead to increased cell wall permeability and, as a result, the death of the bacteria. In P. aeruginosa, polymyxin resistance is strictly associated with covalent addition of 4-amino-L-arabinose to phosphate groups within the lipid A and core oligosaccharide moieties of lipopolysaccharides. Genes in the arnBCADTEFpmrE operon encode enzymes responsible for synthesis and transfer of L-Ara4N to lipid A. This amino-sugar modification interferes with charge interactions between phosphate groups within lipopolysaccharides and amino groups within the cyclic polymixin oligopeptide. In Gramnegative bacteria (including P. aeruginosa) the PmrAB and PhoPQ two-component regulatory systems stimulate transcription of the arnBCADTEF-pmrE operon in response to antimicrobial peptide exposure or divalent cation depletion. The sensor kinase PmrB activates the transcriptional response regulator PmrA through a phosphotransfer relay. Activation of PmrA can also occur as a consequence of pmrB mutation, previously observed as a cause of polymixin resistance in a laboratory strain as well as in clinical isolates [64].

Plasmid is most commonly a fragment of double-stranded circular DNA present in bacterial cells, physically separated from chromosomal DNA, which can replicate independently of the chromosome and has the ability to move to other cells. Some of the genes carried by the plasmid have a beneficial role for the host cells. One such example is enabling the host cell to survive in an environment that would be lethal or restrictive for growth. Some of these genes encode traits for antibiotic resistance as well as resistance to heavy metals [23, 55]. These resistance plasmids can be transferred from cell to cell through the mechanisms of transformation, conjugation and transduction. Conjugation is a very rare phenomenon, probably because it involves strict requirements. The donor cell must contain a conjugative element (it can be transposon or plasmid), and donor as well as recipient cells must establish physical contact that is sufficiently stable to allow the transfer of DNA. Both cells must be metabolically active to allow DNA synthesis (Fig. 3). The transduction process requires a metabolically active donor cell in which transducing phage particles are produced during viral reproduction. The recipient can be spatially and temporarily separated from the donor, because the genetic information in the transducing particle can persist. In the transduction process the resistance gene can be transferred by phages because they are often resistant to many physical and chemical agents and can survive in the environment. The transformation process does not require a living donor cell, because the release of DNA during death and cell lysis is sufficient to provide free DNA. The persistence and dissemination of DNA in the environment determine how far in time and space the recipient cell can be separated from the donor. The recipient must be physiologically active to be able to adopt DNA. A close genetic relationship between donor and recipient cells is not necessary for the transfer of genes by transformation [14, 55, 93].

Fig. 3.

Fig. 4.

Studies of multiple antibiotic resistance determined by plasmids and transposons in Gram-negative bacteria (including Pseudomonas spp.) lead to the discovery of integrons. Integrons are genetic elements that represent an excision – an integration system involved in the capture, mobilization, and expression of gene cassettes conferring resistance to β-lactams, aminoglycosides, chloramphenicol, trimethoprim, sulfonamides, spectinomycin, streptomycin, and other antibiotics. All known integrons possess three elements essential for capturing exogenous genes: the integrase (intI) gene, a primary recombination site (attI), and a strong promoter. Gene cassettes are captured with the use of attI which allows recombination between the attI of the integron and a 59-nt element (also known as attC site) of a cassette. Unlike other excision-integration systems, integrons have multiple tandem gene cassettes inserted in one attI site and intI adjacent to attI rather than located in a gene cassette [43].

Aminoglycosides are commonly used in the treatment of infections caused by P. aeruginosa such as tobramycin, gentamicin, amikacin [79]. The mechanism of bacterial resistance to aminoglycoside antibiotics is very complicated and can run in three directions. In the first mode, the antibiotic can be subjected to enzymatic modification, the structure of the cellular receptor may change and the membrane permeability and transport of the antibiotic into the interior of the cell are disturbed [80, 83]. The second mechanism of bacterial resistance to aminoglycosides (characterized especially for Gram-negative bacteria, including P. aeruginosa) is based on the disruption of the energy-dependent antibiotic transport system to the cell receptor (ribosome 30S subunit), as a result of which the antibiotic does not reach the target site of action [80].

The third mechanism is based on a chromosomal mutation and leads to a change in the S12 protein of the ribosome 30S subunit and the loss of antibiotic binding ability [23]. Bolard et al. [98] documented that resistance of clinical strains of P. aeruginosa to aminoglycosides resulted from the production of trans ferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and mutational derepression of intrinsic multidrug efflux [98].

Fluoroquinolones have a broad range of antibacterial activity which is useful in the treatment of different diseases (e.g., urinary tract infections, gastrointestinal infections, respiratory tract infections, sexually transmitted diseases, bone and joint infections, and infections of the skin and soft tissue) [80]. The fluoroquinolones resistance genes are located in bacterial chromosome and mutations are the main mechanism of this resistance. The resistance to these antibiotics is predominantly caused by mutations in DNA topoisomerase II (gyrase) and topoisomerase IV enzymes [79]. These genes are responsible for the introduction and/or removal of supercoils in and the catenation/decatenation of DNA. Thus, they play an essential role in the replication, transcription, recombination, and repair of DNA. Gyrase is the preferred target of fluoroquinolones in Gram-negative bacteria; therefore, the resistance mutation first occurred in this enzyme. In some highly resistant isolates, additional mutations in topoisomerase IV can be observed. Both gyrase and topoisomerase are each comprised of two subunits, with fluoroquinolones resistance mutations occurring typically in the region assigned as “quinolone resistance determining region” (QRDR) of GyrA and/or ParC. Such mutations commonly occurred in fluoroquinolones-resistant P. aeruginosa [79].

An active efflux of the agents via antibiotic efflux pump is a typical phenomenon in P. aeruginosa where efflux-mediated resistance to quinolones (along with mutations in the genes of gyrase and topoisomerase) seems to predominate as a mechanism of resistance to these agents. The active efflux mechanism is due to the broad substrate specificity of the fluoroquinolones efflux systems, which are capable of accommodating a variety of clinically relevant antimicrobial agents in addition to them [80]. Four members of the Resistance Nodulation Division family of multi-drug efflux systems, MexAB-OprM, MexCD-OprJ, MexEF-oprN, and MexXY-OprM are described to accommodate fluoroquinolones. Expression of mexAB-oprM, controlled directly or indirectly by three repressors MexR, NalD and NalC and mutations in mexR, nalC and nalD, have been observed in fluoroquinolone-resistant clinical P. aeruginosa isolates [79].

Besides fluorquinolones, β-lactam antibiotics are most frequently applied in the treatment of bacterial infections (e.g, bacterial meningitis, otitis media, salmonellosis). During the past 30 years, emergence and dissemination of β-lactam resistance in some pathogenic strains such as P. aeruginosa has become a serious problem worldwide. Of particular concern is the increasing resistance to 3^rd and 4^th cephalosporins generation and carbapenems. Gram-negative bacteria pursue various molecular strategies for developing resistance to these antibiotics, most commonly the ability to hydrolytic cleavage of the β-lactam ring of cephalosporins by β-lactamases, antibiotic expulsion by chromosomally encoded efflux mechanisms and reduced drug uptake owing to the loss of outer membrane porin proteins [79]. In P. aeruginosa four molecular classes (A-D) of β-lactamases, including metal dependent (Zn²⁺ – equiring, class B) and metal-independent (active site serine, classes A, C, and D) β-lactamases have been observed (79). P. aeruginosa typically carries chromosomal genes for two of the β-lactamases. It acts as a class C cephalosporinase (AmpC) and a class D oxacillinase (PoxB) [79]. AmpC is a well-known β-lactamase while PoxB activity was only detected in lab mutants lacking AmpC. AmpC is induced by a number of β-lactam antibiotics; therefore, it contributes to the internal resistance to the number of β-lactam antibiotics. However, AmpC cannot be induced by monobactams, the anti-pseudomonal penicillin piperacillin and many of the newer cephalosporins such as cefotaxime, ceftriaxone, ceftazidime and then the resistance is dependent upon mutational derepression of ampC (in fact, mutational derepression of ampC is the most common mechanism of resistance to β-lactams in P. aeruginosa) [79].

The original β-lactamases were plasmid-encoded class A enzymes with limited-spectrum of activity that could only hydrolyze penicillin and an older narrow-spectrum of cephalosporins. However, there are reports in the literature about the presence of acquired β-lactamases in P. aeruginosa with an extended-spectrum of β-lactamase (ESBL) enzymes (classes A and D) capable of hydrolyzing a broader range of β-lactams, including a broad spectrum of cephalosporins, monobactams and carbapenemases (classes A, B and D) that hydrolyze most β-lactams, including carbapenems (excluding aztreonam) [76, 79].

Because bacteria produce effective resistance mechanisms against antibiotics commonly available and in use, and in addition, this resistance can be passed between them, the focus should turn to a quest for alternative methods of inhibiting bacterial metabolic activity. If, however, in some cases chemical antibiotics could be replaced by natural substances, the phenomenon of antibiotic abuse could be overcome and the spread of the antibiotic resistance phenomenon may be stopped or at least slowed down. Plant oils are an example of such natural bacteriostatic substances. There are currently many in vitro studies on the mechanisms of their antimicrobial activity.

Many plant EOs are known to inhibit the growth of Pseudomonas strains [e.g., 3, 27, 31, 48, 56, 67, 68, 74, 75, 82, 87, 94, 96]. The antibacterial activity against some Pseudomonas species of oregano, rosmarinus, thymus [1], satureja [16, 74, 75] eucalyptus [74], basil, citrus, fennel, lemongrass [75] has been confirmed. Examples of the minimal inhibitory concentrations (MIC) of selected EOs with biostatic activity against bacteria from Pseudomonas genera are collected in Table I. A comparison of the value of MICs is impossible, because the authors used different units and in most cases the data needed to re-calculate such values are missing. However, the MIC values are generally very low. That ability of EOs to inhibit bacteria grow in low concentrations is the main advantage of using them. According to literature data, the exposure of cells to such concentrations of natural antimicrobial agents neither kills bacteria nor stops their growth and thus does not cause selective pressure or the development of resistance mechanisms in bacteria cells [74].

The mechanism of antimicrobial activity of EOs is closely related to the type and amount of antimicrobial-active compounds in their composition. The active ingredients of EOs are a diverse group of organic compounds with a low molecular weight [12, 34, 40, 45]. The composition of the essential oil of a given plant may vary depending on the part of the plant from which the oil was obtained (leaves, flowers, etc.), the location and conditions of cultivation, and methods of isolation [12, 33, 45]. Due to the variability of the composition of EOs, the mechanism of their action is still not fully understood [34, 40, 48].

Pseudomonas bacteria are particularly resistant to antibiotics and disinfectants because of the increased coherence of the cytoplasmic membrane, due to the higher content of lipoproteins [44, 95]. Ingredients of EOs are characterized by high lipophilicity, thanks to which they have the ability to penetrate the membrane and cell wall of microbial cells. These molecules, after penetrating the cell, disrupt its integrity [34, 45]. It should be noted that the components of EOs can eliminate bacterial plasmid antibiotic resistance by inhibiting the replication of a given plasmid, thanks to which they can also exhibit antimicrobial activity against strains resistant to previously used substances [45]. Unfortunately, several factors still limit the effectiveness of EOs on bacterial cells. It has been shown that the penetration of essential oil components into Gram-negative bacteria cells is a more complicated process than in the case of Gram-positive bacteria due to differences in the structure of their cell walls [10, 12, 34, 45]. Differences in cell structure between bacterial species and strains also determine the potency of the antimicrobial activity of a given essential oil. It has been proved that the formation of exopolysaccharides occurring in Pseudomonas species causes their greater resistance to EOs than other Gram-negative bacteria [45].

The ingredients of EOs, due to their hydrophobicity, can affect the proportion of unsaturated fatty acids in the cell membrane and change their structure. The use of concentrations of oils lower than the MIC value in relation to bacteria can cause an increase in the amount of unsaturated fatty acids responsible for the fluidity of the cell membrane, which results in structural changes of the membrane [45]. Phenolic compounds contained in EOs also have a significant influence on their antibacterial activity. Treatment with thymol, carvacrol and eugenol may increase the content of saturated fatty acids while lowering the unsaturated fatty acid content in the bacterial cell membrane. This results in an increased stiffness of the cell membrane and eventually its degradation. Essential oil components may also affect the activity of enzymes responsible for the synthesis of fatty acids included in the outer layer of the cell [10, 12, 22].

Active substances in EOs can affect proteins in bacterial cells and affect cell division. These compounds also affect the expression of genes. They can affect the regulation and synthesis of, for example, enzymes or chaperone proteins, inter alia related to the thermal stress of the cell. The action of essential oil components in relation to cellular proteins completely disturbs the functioning of the bacterial cell [10, 12, 40, 45].

The production of ATP in bacterial cells takes place in the cytosol by glycolysis. There is a correlation between intracellular and extracellular ATP concentration. The ingredients of EOs cause permeabilization of the cell membrane, thereby reducing the concentration of ATP inside the cell. Permeabilization of the cell membrane and leakage of ions (e.g. hydrogen and potassium) also causes excessive loss of inorganic phosphate, resulting in a reduction in the amount of intracellular ATP. The ATP content in the bacterial cell may also decrease due to the inhibitory activity of the oil components towards ATP-ase [10, 18, 40, 45, 48, 70]. EOs can affect bacterial cell metabolites [10, 40, 92, 95]. This effect may take place by interacting with metabolites already present in the cell or by altering metabolic pathways. This results in changes in the function of certain metabolites, as well as the synthesis of excessively small or large amounts of specific compounds. Changes in metabolic pathways may also result in the formation of new metabolites. This causes the occurrence of cellular stress and significant disturbances in the functioning of the cell [45].

Fig. 5.

EO components have the ability to modify the morphology of a bacterial cell. It has been shown that oblong-shaped cells are more sensitive to these compounds than coccoid ones. Changes in bacterial morphology are also different for Gram-positive and for Gram-negative bacteria. The action of EOs on bacteria can cause changes in the shape of cells, for example by extending or rounding them. It can be observed that bacterial cells treated with oils cease to be visibly separated from each other due to the degradation of their cell membranes. Damage to the protein-lipid membrane can lead to coagulation of the cytoplasm and a complete release of the cellular content. Changes of this type involve bacterial cell lysis [10].

The compounds included in the EOs are able to inhibit the phenomenon of Quorum Sensing (QS), which is an intercellular communication system occurring at a sufficiently high cell density. QS regulates a number of activities, such as biofilm formation, sporulation or virulence factors [19]. The expression of genes involved in QS results in the production of signalling molecules. The inhibition of the phenomenon by the components of oils takes place by inhibiting the synthesis, transport or secretion of signalling molecules. This leads to a reduction in the formation of bacterial biofilms, proteolytic activity and to the reduction of bacterial virulence [48].

EOs have multidirectional antibacterial activity, which is associated with the diversity of their composition. Each of the biologically active compounds present in the essential oil has a different mechanism of action. There is a synergy between the compounds, which makes the oils work more comprehensively than all their components separately. In general, the activity of each essential oil consists of a series of activities of individual substances or their complexes, which results, inter alia, in decreasing cellular respiration and lowering intracellular pH, and eventually may lead to the inhibition of bacterial cell growth or lysis [10, 12, 40, 45].

Although there are many scientific reports documenting (in vitro) the strong antibacterial activity of essential oils against Gram-negative and Gram-positive bacteria, there are no reports from in vivo experiments. We can only find the information that some plant oils/extracts are used in the folk medicine in Brazil to treat some bacterial infections [38]. Unfortunately, there is no data available on its form of application and we do not know the concentration of oils contained in natural medicines and how effective they are.