re-Evaluating the Synergistic Potency of Essential Oils and Antibiotics Against Klebsiella pneumoniae BLSE Strains

ABSTRACT


Introduction
Since the discovery of penicillin in 1928, antibiotics have greatly contributed to the reduction of mortality from infectious diseases. However, the excessive, frequent, and uncontrolled use of antibiotics in human and animal health has led to the emergence and spread of bacterial resistance over time. Antibiotic resistance is now a major public health issue (1). Klebsiella pneumoniae, a Gram-negative bacillus of the Enterobacteriaceae family, is known to be highly virulent and multidrug-resistant. It is an opportunis-tic pathogen that causes respiratory tract infections, such as pneumonia, sepsis, and urinary tract infections (2). In 2017, this species was placed on the World Health Organization's Priority 1 (Critical) list, as an urgent research target for the development of new antibiotics. The evolution of K. pneumoniae strains, through point mutations and the horizontal transfer of mobile elements, together with the misuse of ß-lactam and other antibiotic families, has led to extended-spectrum ß-lactamases (ESBLs) and carbapenemases being produced and rapidly disseminated. Studies have shown that enzymatic mechanisms of antibiotic re-sistance are the most common in K. pneumoniae (3). Recently, resistance to fosfomycin, carbapenems, tigecycline, and newer combinations such as meropenem-tazobactam, ceftazidime-avibactam, and ceftolozane-tazobactam have been reported around the world (4,5). The emergence of antibiotic-resistant strains and their spread across the globe necessitates research into the development of new molecules with natural antibacterial activity (6). Essential oils (EOs) have long been known for their antimicrobial properties and can act on the defense systems of cells, such as efflux pumps, quorum sensing, biofilm formation, and ATPase pump inhibition (7). The main components of EOs with antimicrobial activity are terpenes, alcohols, acids, esters, epoxides, aldehydes, ketones, amines, and sulfides such as terpineol, thujanol, myrcenol, neral, thujone, camphor and carvone (8,9). Several studies have demonstrated the effectiveness of EOs against multidrug-resistant K. pneumoniae strains, including ESBL and CTX-M positive strains (10,11). Combining antibiotics with EOs represents a novel exploration in the search for bioactive molecules with anti-microbial effects. The synergistic effect between these molecules could be a viable alternative to using antibiotics alone and help to overcome the problem of antibiotic resistance in therapy (12). Eucalyptus globulus EO possesses antimicrobial properties against various pathogenic bacteria such as Mycobacterium tuberculosis, Staphylococcus, and gram-negative bacteria like K. pneumoniae. The activity is mainly attributed to components like 1,8-cineole, p-cymene, limonene, β-pinene, and α-terpinol (13). Moreover, Syzygium aromaticum is rich in aromatic constituents, and its EO exhibit antioxidant, antifungal and antimicrobial activities due to the majority compound eugenol (14). Species from the Lamiaceae family are known for their therapeutic effects, and this includes Thymus algeriensis, a thyme species found in North Africa (Algeria, Libya, Morocco and Tunisia). Its EOs have various medicinal properties such as antimicrobial, antioxidant, anticancer, anti-ulcer, anti-diabetic, insecticidal, and anti-inflammatory activities (15). Our study aimed to first determine the chemical composition of the EOs of three plants two of them growing wild in Tizi-Ouzou region, namely T. algeriensis and E. globulus. For S. aromaticum, we used cloves directly purchased from an herbalist. We then conducted a study of their antibacterial activities and a second study of the association of these three EOs with antibiotics against clinical strains of K. pneumoniae ESBL and CTX-M positive. The main scope of this study was to explore the potential synergistic effects of combining antibiotics with the three EOs on these clinical strains. By assessing the efficacy of this combination, we hoped to discover if one of these EOs could enhance the efficacy of antibiotics against this strain of bacteria.

Plants and extraction of essential oils
Two plants were collected from the Tizi-Ouzou region in the locality of Aghrib (36° 48'08'' North 4°19'22'' East) for T. algeriensis, and from Yakouren (36°44'05'' North 4°26'19''East) for E. globulus. S. aromaticum was procured from a local herbalist in Tizi-Ouzou. The identification of the plants was done by Dr. Hocine Abbaci from the University of Bejaia. To extract the EOs, dried leaves were subjected to steam entrainment for 3 hours, after which the EO was recovered by decantation and dehydrated with anhydrous sodium sulfate. All pure EOs were stored at +4°C until use. The EOs yields were 1.8%, 2.5%, and 5.2% for E. globulus, T. algeriensis, and S. aromaticum, respectively.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The chemical composition of EOs was carried out at the Center for Physico-Chemical Analyses (CRAPC) in Tipaza, Algeria. GC-MS analyses were performed using a Hewlett Packard Agilent 6890 plus device equipped with an HP-5MS column (30 m, 0.25 mm, 0.25 μm) and a Hewlett Packard Agilent 5973 mass detector operating in ICT Scan mode (30 to 550). Helium was used as a carrier gas at a flow rate of 0.5 ml/min. The oven temperature program was as follows: 60°C for 8min, 2°C/min up to 250°C., and isothermal for 10min. The detector temperatures were 280°C. The constituents identification was based on comparing their relative retention indices (RI) and mass spectra with those of reference constituents in the NIST05 library or with mass spectra in the literature (16). The relative amount of the individual components of the total oil was expressed as a percentage of the peak area relative to the total peak area.

Tested Bacterial Isolates
Seven bacterial strains were utilized: three reference strains -Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumoniae ATCC 700603 ESBL-positive (SHV-18) and four clinical strains of multidrug-resistant ESBL and CTX-M positive, K. pneumoniae 3520, K. pneumoniae 1216, K. pneumoniae 5111 and K. pneumoniae 3511. These strains were isolated from urine and pus samples at the Tizi-Ouzou University Hospital Microbiology-Parasitology Laboratory. Identification with MALDI-TOF and the search for resistance genes were conducted at the Laboratory of Molecular Microbiology, CHU ULB-ERASME, Belgium. The strains were then stored at -18 °C, revived on their selective media, and grown on heart-brain broth.

Antibacterial activity of essential oils
A disc diffusion method was used to study the antibacterial activity of our EOs. Muller-Hinton Agar was seeded with a suspension of the target bacterium at 10 8 CFU/mL. Whatman No.1 paper discs with a 6mm diameter were placed on the bacterial mat and loaded with 10μl of pure EO. The boxes were then pre-diffused in the agar by storing them at 4 °C for 3 hours. After 24 hours of incubation at 37°C, the inhibition diameters were measured. Each test was repeated three times. The inhibition diameters were categorized as follows: Ø < 8mm as non-sensitive (-); 8mm < Ø< 14mm as sensitive (+); 14mm <Ø < 20mm as very sensitive (++) and finally Ø > 20 mm as extremely sensitive (+++). A Chloramphenicol disc (30μg) was utilized as a positive control (17).

Determination of minimal inhibitory concentration
The minimum inhibitory concentration (MIC) of EOs against ESBL strains of K. pneumoniae was determined using the 96-well microplate microdilution method in broth according to the Clinical and Laboratory Standards Institute (CLSI). Semi-logarithmic dilutions of T. algeriensis, Tukey's HSD (honestly significant difference) test at p < 0.05. All statistical analyses were performed using SPSS Statistics (Version 25.0).

Screening the Synergistic Effect of the EO with Antibiotic Discs
Tables 3, 4, and 5 in this study demonstrate two types of interactions between the three antibiotics and three EOs: antagonism and synergy. Specifically, a synergy-like interaction was observed between amoxicillin-clavulanic acid and T. algeriensis EO on two pathogenic strains (KP 1216 and KP 3511) and the reference strain K. pneumoniae ATCC 700603 ESBL-positive. Compared to the amoxicillin-clavulanic acid disc alone, the IZD of the combined disc increased from 2 to 4mm, indicating a synergistic effect. The Mann-Whitney test showed a significant difference (p < 0.05) in the antibacterial activity of the combined disk (amoxicillin-clavulanic acid-T. algeriensis EOs) and the single disks of amoxicillin-clavulanic acid and T. algeriensis EOs , which were tested, against the K. pneumoniae 3511, K. pneumoniae 1216 and K. pneumoniae ATCC 700603 strains.

Checkerboard method
The checkerboard method was used in this study to E. globulus, and S. aromaticum EOs (433.5-0.84mg/ml), (441-0.125mg/ml) and (512-1mg/ml), respectively, were prepared in Muller-Hinton broth with tween 80 at a final concentration of 1% (v/v). Each well was then filled with 50μl of EO dilution and 50μl of the bacterial suspension at a concentration of 10 6 CFU/ml. A growth control was then performed with Muller-Hinton broth alone and with tween 80 at 1% to exclude the antibacterial effect of tween 80. After being incubated for 18h at 37°C, 30 μl of resazurin at 0.015% was added to each well and incubated at 37°C for 3 hours. The MICs of the EOs were determined by noting the first blue, unchanged well after 3 hours of incubation with resazurin, and expressed in mg/ml.

Screening the Synergistic Effect of the EO with Antibiotic Discs
Three antibiotics Amoxicillin-clavulanic acid (20/10μg) (AMC); Ceftazidime (30μg) (CAZ) and Levofloxacin (5μg)(LEV) (Liofilchem brand) were tested alone and in combination with three EOs on Mueller Hinton agar seeded with 10 8 CFU/ml. 10μl of each EO was impregnated onto the antibiotic discs. After 24 hours of incubation at 37°C, the diameters of the inhibition zones around the discs of the EOs alone, antibiotics alone, and discs combined antibiotic-EOs were measured. The results were then analyzed for antagonism and synergy by comparing the diameters of inhibition in combination (EO + antibiotic) with the sum of the diameters of the inhibition zones of the two agents tested separately (EO and antibiotic) (18).

Statistical Analysis
The data from synergetic tests between antibiotics and EOs were analyzed using the Mann-Whitney's HSD (honestly significant difference) test at p < 0.05 to investigate differences between single molecules and combination groups. Three independent experiments were performed to carry out all antimicrobial analyses, and the results were presented as the mean ± standard deviation and were subjected to the one-way ANOVA followed by confirm the synergy observed on a solid medium between the disc of Amoxicillin-Clavulanic Acid and the EO of T. algeriensis. The results, depicted in Table 6, indicate an Inhibitory Fraction below 0.5 for the three strains tested: K. pneumoniae 3511, K. pneumoniae 1216, and K. pneumoniae ATCC 700603, demonstrating a synergistic effect between the EO and Amoxicillin-Clavulanic Acid. The MICs for Amoxicillin-Clavulanic Acid decreased by 25% in K. pneumoniae 1216 and 12.5% in K. pneumoniae 3511 and K. pneumoniae ATCC 700603, respectively. The Mann-Whitney test showed significant differences (p < 0.05) in the antibacterial activity expressed as MIC of the combination of amoxicillin-clavulanic acid-EO of T. algeriensis and the MIC of amoxicillin-clavulanic acid tested alone against the strains K. pneumoniae 3511, K. pneumoniae 1216 and K. pneumoniae ATCC 700603. The decrease in the MIC of amoxicillin-clavulanic acid in combination with EO of T. algeriensis is statistically significant.

Discussion
Plants are subjected to biotic and abiotic stresses that influence their physiology and growth. In order to resist     (37) found that eucalyptol or 1.8-cineole can increase the zeta potential and membrane permeability of K. pneumoniae KPC positive strains, resulting in a loss of intracellular proteins and nucleic acids, as well as an increase in oxidative stress and the peroxidation of membrane lipids. Linalool, the second major compound in the EO of thyme, also acts on K. pneumoniae KPC strains by inducing the loss of cytoplasmic and membrane proteins (30). Similarly, the hydroxyl group present on the C1 of the mono-terpene ring in thymol is responsible for its activity against pathogenic bacteria such as S. aureus, P. aeruginosa, and E. coli (38). Eugenol, a major constituent of the EO of S. aromaticum, has been reported to act on resistant strains of K. pneumoniae BMR and carbapenem by decreasing intracellular ATP concentration, intracellular pH, and hyperpolarizing the plasma membrane, thus increasing its permeability (39 (46) demonstrated the inhibition of CTX-M ESBL by methyl cinnamate, a major compound found in the EO of Ocimum basilicum. The mechanisms of action behind the synergistic or additive effects of combining EOs and antibiotics are still not fully understood. While membrane destabilization is the most commonly studied mechanism, it cannot explain all the observed effects. Therefore, it is premature to speculate about the future of these formulations, and further research is needed to evaluate their stability, in vivo efficacy, and toxicity. However, these combinations have the potential to address the issue of antibiotic resistance by allowing the use of lower doses of antibiotics that may have been abandoned due to side effects or resistance. EOs can also destabilize the resistance mechanisms of bacteria, making these combinations a promising approach to combat bacterial infections.

Conclusion
K. pneumoniae producing ESBL poses huge therapeutic challenges due to its multidrug resistance. Research is needed to reduce the spread of resistance, identify new ways to treat infections, and gain a better understanding of resistance mechanisms. In this context, we assessed the chemical composition of T. algeriensis, S. aromaticum, and E. globulus EOs and tested them against K. pneumoniae ESBL strains. GC-MS identified thymol, linalool, and p-cymene as the main components of T. algeriensis; eucalyptol, α-pinene, aromadendrene, and pinocarveol in E. globulus; and eugenol and eugenol acetate in S. aromaticum. Results of the activity tests showed that all three EOs were active against the tested strains. The interaction tests showed a synergistic effect between amoxicillin-clavulanic acid and T. algeriensis EO on two K. pneumoniae ESBL strains. These findings suggest the potential of our EOs to inhibit multi-resistant pathogenic bacteria, as well as their synergistic interaction with antibiotics, which could be an alternative to antibiotic use alone.

Funding
No Funding.

Institutional Review Board Statement
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Informed Consent Statement
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Data Availability Statement
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