Antibacterial activity of Centaurea pumilio L. root and aerial part extracts against some multidrug resistant bacteria

Background In the context of searching for potent, safe, natural antimicrobial agents to combate the global antimicrobial resistance (AMR) phenomenon, the current study evaluates for the first time ever, the broad-spectrum antimicrobial activity of essential oil (EO) and extracts from the rare wild plant Centaurea pumilio L.. It has tremendous ethnomedicinal values; its dried root is used as a fattening agent, a treatment for bad breath and diabetes, and screened for schistosomicidal activity. Methods C. pumilio EO was extracted by hydrodistillation using a Clevenger apparatus. Chemical constituents of aerial part were extracted using a sequential solvent/solvent procedure employing four solvents with increasing polarities in the following order: petroleum ether, chloroform, ethyl acetate, and n-butanol. The chemical constituents were identified by GC-MS. Fifty-two microbial strains were used; twenty-six multidrug resistant (MDR), sixteen clinical, and ten reference strains. The identification of the microbial strains was performed by MALDI-TOF-MS. The antimicrobial activity of the EO and the aerial part and the root extracts was assessed through disc diffusion assay. A minimum inhibitory concentration (MIC) of the EO and extracts was determined using the broth micro-dilution method. Results The growth of reference and clinical strains was inhibited by EO, methanol, chloroform, and ethyl acetate aerial part extracts and chloroform root extract. The MDR strains growth, however, was inhibited only by EO and chloroform aerial part extract. GC-MS identified for the first time eighteen constituents from aerial part EO and chloroform extract each. EO showed antimicrobial activity against the reference, clinical, and MDR strains with MIC values of 31.25–125, 31.25–125, and 62.50–250 μg/mL, respectively. Methanol aerial part extract exhibited high antimicrobial activities with MIC values of 62.50–250 μg/mL against reference and clinical strains. Chloroform root extract displayed strong antimicrobial activity against reference and clinical strains recording MIC values of 62.50–250 μg/mL and 62.50–125 μg/mL, respectively. The chloroform aerial part extract demonstrated potent antimicrobial activity against the reference, clinical, and MDR strains with 31.25, 31.25, and 15.62 μg/mL MIC values, respectively. Conclusions Present data unravel the C. pumilio pharmacological magnitude to discover eco-friendly potent antimicrobial agents to fight AMR phenomenon.


Background
Antimicrobial resistance (AMR), a life-threatening and multifaceted global phenomenon, is a consequence of improper and/or overuse of antibiotics [1]. The Centre for Disease Control and Prevention (CDC) and the World Health Organization (WHO) report alarmingly increasing mortality rates as a result of infections from various MDR strains [2]. Some of the most lifethreatening MDR strains with severe human implications worldwide are Staphylococcus aureus MRSA, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumonia [3]. Globally, intensive care units (ICUs) are considered foci for the proliferation and promotion of persistent infection with MDR strains [4]. In Egypt, several studies have been conducted to trace the incidence and prevalence of AMR and its possible reasons, especially in ICUs [5,6]. Infection with Gram-negative MDR strains (e.g., Acinetobacter sp., Klebsiella sp., and Pseudomonas sp.) with a higher frequency than Gram-positive MDR strains was reported in the ICUs of Alexandria hospitals [4].
Increased health care expenditure for patients infected with MDR strains is one of the burdens imposed by AMR [7]. The Organization for Economic Cooperation and Development (OECD) reported that resistant microbes currently cause 700,000 deaths annually. If the current trends persist, the number of deaths is expected to rise to ten million by 2050, displacing cancer as one of the prime causes of mortality [8].
To combat AMR, innovative approaches must be adopted in the research for novel antimicrobial medicines. As the use of synthetic chemicals to combat MDR strains is highly restricted because of health and environmental considerations [9], turning to natural products is, then, an excellent alternate to control the prevalence of AMR. Centaurea spp L. are well-known for their bioactive secondary metabolites with antimicrobial potential against Gram-positive, Gram-negative, and MDR bacteria [10][11][12]. They have been reported to possess medicinally important EOs and large numbers of terpenoids with more than 3000 different structures [12]. The antimicrobial activity of many Centaurea spp. L. (e.g., C. pulcherrima, C. consanguinea, C. ptosomipappa, C. chamaerhaponticum, C. amanicola, C. sessilis, C. armena, and C. aladagensis) has already received researchers' attention [12]. Centaurea pumilio, Synonym C.
aegialophila is a rare species that can be found scattered on sand dunes along the Egyptian Mediterranean coast [13]. Its dried root is frequently used as a fattening agent in traditional Egyptian medicine [14] and the indigenous people commonly use the peeled root to treat diabetes and bad breath. It has also been screened for efficacy against schistosomiasis and showed antioxidant activity [14]. C. pumilio is regarded as an endangered plant and has been included on the national Red List of the International Union for Conservation of Nature (IUCN) of threatened plants [13].
The objective of the present study was to extract EO and other active volatile constituents from the aerial part and the root of C. pumilio in order to assess the antimicrobial potential against 26 MDR strains collected from ICUs, 10 reference, and 16 clinical strains. To the best of the authors' knowledge, the current work represents the first study to separate and assess the antimicrobial potential of the active constituents in C. pumilio.

Reagents and chemicals
The chemicals and reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Plant materials
The plant was collected from its natural habitats on the Northern coast of Egypt during their flowering season (April and May 2018). It was identified as Centaurea pumilio L. by Prof. Dr. Salama El-Darer (Botany Department, Faculty of Science, Alexandria University, Egypt). A voucher specimen (CP019) was deposited in the herbarium of the Pharmacognosy Department. The fresh plant (Additional file 1) was air-dried, placed in a tightly sealed container, and stored in a cold, dark, dry place until the analyses were carried out. Permission to collect the plant samples was not required.

Preparation of C. pumilio EO and extracts
The EO was extracted in accordance with previously reported procedures [15]. The air-dried aerial part (400 g) was subjected to hydrodistillation using a Clevenger apparatus. The resultant pale yellow oil (Additional file 2) was stored at − 20°C. Meanwhile, a solvent/solvent extraction method was employed to extract the active constituents from the aerial part and the root [16]. The powdered air-dried aerial part and root of C. pumilio (700 g) were extracted separately with 4 L methanol (70%) until exhaustion. Both methanol extracts were filtered and concentrated under vacuum. The aqueous solutions were further extracted sequentially using four solvents with increasing polarities in the following order: petroleum ether, chloroform, ethyl acetate, and n-butanol. The organic phases of each extract from the aerial part and root, and the remaining aqueous extracts, were evaporated under reduced pressure (Fig. 1a, b).

GC-MS analysis
The gas chromatography-mass spectrometry (GC-MS) analyses of C. pumilio EO and chloroform aerial part extract were carried out using a GC-MS instrument with the following specifications: a TRACE GC Ultra Gas Chromatographs (Thermo Scientific Corp., USA) coupled with a Thermo mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer) [15,16]. The experimental conditions of the GC-MS system were as follows: TR-5MS capillary standard non-polar column, dimension: 30Mts, ID: 0.25 mm, and film thickness: 0.25 μm. The flow rate of the mobile phase (carrier gas: He) was set at 1.0 ml/min. In the gas chromatography phase, the temperature program (oven temperature) was set at 40°C and then raised to 250°C at 5°C/min and the injection volume was 1 μL. The identification of the chemical constituents was de- convoluted using AMDIS software (Available at http:// www.amdis.net) and by their retention indices (RI determined with reference to homologous series of n-alkanes C 9 -C 40 , under identical experimental conditions), MS library search (NIST 08 MS Library Version 2.0 f), and WILEY MS 9th Edition (Thermo Fisher Scientific Austria) and by comparing with the MS literature data.
The relative amounts of individual components were calculated based on the GC peak area (FID response) without using a correction factor.

Standard commercial antibiotics
Thirty-six commercial antibiotics (Sigma-Aldrich, St. Louis, MO, USA) were employed to assess the antibiotic resistance profile of the MDR strains. They were supplied as discs with known concentrations and are fully described in Additional file 3.

Determination of antimicrobial activity Bacterial strains
In the present study, ten reference strains:

Disc diffusion assay
The antibiotic resistance patterns of the MDR strains and the antimicrobial potential of the EO and extracts from C. pumilio against all the strains tested (i.e., reference, clinical, and MDR strains) were determined using the Kirby-Bauer disc diffusion technique [17,18] as described by CLSI guidelines. An inoculum of 0.5 McFarland was used to inoculate the MHA and SDA agar plates. The EO or plant extract (20 μL) was loaded onto the sterile filter paper discs at a concentration of 100 mg/ mL (2 mg/disc). Filter discs containing standard antimicrobial agents (ampicillin and fluconazole), EO, and extracts were employed in the pre-inoculated agar plates. The negative control plates were performed using dimethyl sulfoxide (DMSO). The diameters of the inhibition zones were measured and all the experiments were conducted in triplicate.

Determination of minimal inhibitory concentration
The minimum inhibitory concentrations (MIC) of the EO and extracts were determined with the broth microdilution method [18]. This was performed with 96-well microtiter plates using resazurin dye [19]. One hundred microliters (100 μL) of cell suspensions (

Statistical analysis
Data were statistically expressed in terms of means (n = 3) ± standard error (SE). Statistical analysis was run by the STATISTICA 10 of StatSoft, Inc. (2011) (Tulsa, Oklahoma, USA) [20]. Data variability was checked by one-way ANOVA at P < 0.05 for plant extracts and a standard antibiotic against strains (indicated with capital letters (A-E) in the same row). Factorial ANOVA was checked at P < 0.05 for each plant extract and the standard antibiotic to examine the factorial interaction between the reference and clinical strains and Grampositive bacteria and Gram-negative bacteria (indicated with the small letters (a-c) in the same column). The data variability of the chloroform extract from the aerial part was checked by one-way ANOVA at P < 0.05 against the MDR strains; as indicated by the small letters (a-c) in the same column.

Antibiogram of MDR strains
The phenotypic profile of the 26 MDR strains against 36 commercial antibiotics is depicted in Additional file 3. There was a discrepancy in the antibiogram profile among the 26 MDR strains belonging either to the same species or different genera in terms of the number and group of the antibiotics. The uppermost MDR strains exhibited resistance against 10-16 antibiotics.
Antimicrobial activity of C. pumilio extracts and EO against reference and clinical strains The antimicrobial activities of C. pumilio EO and extracts against reference and clinical strains were assessed (Table 3). Using the factorial ANOVA at P < 0.05, the inhibition zone diameters (mm) of the chloroform, the ethyl acetate extracted from the aerial part, and the standard antibiotic showed significant data variability between the reference and clinical strains and between the Gram-positive and Gram-negative bacteria and their interactions (categorical factors). Statistical analysis proved that the highest significant data variability was observed between the Gram-positive and Gram-negative bacterial strains (F = 3.374, P < 0.01) compared to those of the reference and clinical strains (F = 3.744, P < 0.05) and their interactions (F = 2.176, P < 0.05). No significant data variability was observed for the 70% methanol extract from the aerial part, the chloroform root extract, or the EO. The chloroform extract from the aerial part showed the highest inhibition zone diameters compared to the standard antibiotic, other extracts, and the EO. The inhibition zone diameters of the chloroform aerial part extract varied significantly as indicated by the different letters between the reference (mean = 27.11 mm) and clinical strains (mean = 22.42 mm). It also showed significant variance between the Gram-negative (mean = 20.40 mm) and Gram-positive clinical bacterial strains (mean = 26.47 mm). Furthermore, it showed the highest significant variability inferred by one-way ANOVA at P < 0.05 among the other extracts and the standard antibiotic against each strain. The antibacterial activity of the ethyl acetate extract from the aerial part varied significantly between the reference (mean = 16.56 mm) and clinical strains (mean = 15.41 mm), and the Gramnegative (mean = 14.00 mm) and Gram-positive clinical bacterial strains (mean = 18.58 mm).
Antimicrobial activity of C. pumilio extracts and EO against MDR strains Of the four extracts studied, only the chloroform aerial part extract showed potent antimicrobial activity against all the MDR strains tested (Table 4). Using the one-way ANOVA, the chloroform aerial part extract showed significant data variability among the MDR strains tested (F = 3.606, P < 0.05), as indicated by the different letters (a-c). A discrepancy in the antimicrobial potential showed by the chloroform aerial part extract was noted among different strains of the same species. Among the nine MDR A. baumannii tested, inhibition zone diameters ranged from 11.33 ± 0.33 mm to 23.33 ± 0.33 mm. Whilst, among the five MDR tested K. pneumonia, inhibition zone diameters ranged from 12.00 ± 0.00 mm to 25.00 ± 0.00 mm. For the five MDR tested P. aeruginosa and the three MRSA strains tested, inhibition zone diameters ranged from 17.33 ± 0.33 mm to 27.00 ± 0.00 mm and 20.00 ± 0.00 mm to 28.00 ± 0.00 mm, respectively. Conversely, a negligible discrepancy in the range of the inhibition zone diameter was noted among the three MDR E. coli tested.
The growth of only nine out of the twenty-six tested MDR strains was inhibited by the EO (Table 4). At most, the highest inhibition zone diameter was 12.67 ± 0.33 mm. The antimicrobial activity of the EO was not noticed against the nine MDR A. baumannii tested. EO inhibited the growth of only one out of the three MDR E. coli strains tested and only one out of five MDR K. pneumonia strains tested. In contrast, the growth of all the tested MDR P. aeruginosa strains was negatively influenced except for one strain. For the MRSA strains, only two out of three tested strains were adversely suppressed by EO.

MIC of C. pumilio extracts and EO against reference and clinical strains
At most, the highest antimicrobial activity among the four extracts was evidenced by the lowest MIC values observed (Table 5). With regard to the lowest MIC values, the antimicrobial potential of the extracts was in the following order: chloroform aerial part extract >> > chloroform root extract > > methanol aerial part extract = ethyl acetate aerial part extract, when compared to MIC values of standard antibiotics. The chloroform aerial part extract exhibited an MIC value of 31.25 μg/ mL against the Gram-negative bacteria, Gram-positive bacteria, and C.albicans. However, the chloroform root extract showed an MIC value of 62.5 μg/mL against the Gram-negative bacteria and Gram-positive bacteria. At most, the chloroform aerial part extract demonstrated a twofold increase in the antibacterial and anticandidal activity as compared to the standard antibiotic against the clinical strains of K. pneumonia, P. aeruginosa, P. mirabilis, S. enetrica, and C. albicans. Moreover, a four-fold increase in the antibacterial activity of the chloroform aerial part extract was noted against B. pumilus. In contrast, the MIC values of 125-250 μg/mL of the methanol and ethyl acetate aerial part extracts were noticed  against Gram-negative bacteria and Gram-positive bacteria.
The common MIC values for the EO were 62.50 μg/ mL and 125 μg/mL for reference and clinical strains, respectively, against the Gram-negative bacteria and Gram-positive bacteria ( Table 5).

MIC of C. pumilio chloroform aerial part extract and EO against MDR strains
The chloroform aerial part extract revealed its strong broad-spectrum antibacterial activity against the MDR strains with MIC values of 15.62 μg/mL (against 2 MDR strains), 31.25 μg/mL (against 7 MDR strains), 62.5 μg/ mL (against 14 MDR strains), 125 μg/mL (against 2 MDR strains), and 250 (against one MDR strain). Moreover, these MIC values varied greatly among the strains belonging to the same species and among the strains of different species (Table 6)

Discussion
In the ongoing search for natural antibiotics to treat human MDR infections, the current work focuses on looking for natural antimicrobial agents from C. pumilio extracts and EO.
Although the extracts and EOs of the genus Centaurea have been extensively studied and have showed remarkable antimicrobial activity against susceptible and MDR strains [11,12,21], the antimicrobial activity of C. pumilio has not been well explored.
Interestingly, the C. pumilio chloroform aerial part extract exhibited a twofold increase in the anticandidal activity to that of the standard antibiotic. This anticandidal activity is also seen in C. thessala and C. attica chloroform aerial part extracts [33], but not in the chloroform extracts of C. austro-anatolica, C. cariensis subsp. niveo-tomentosa, and C. ensiformis [17,30,34]. Strep. mutans and Steno. maltophilia were inhibited by chloroform extracts of both the root and aerial part of C. pumilio (MIC of 62.50 and 125 μg/mL, respectively). Meanwhile, the C. pumilio chloroform aerial part extract exhibited higher antibacterial activity against MRSA (21, 20, and 28 mm) than that of C. austro-anatolica [17] and C. cariensis [30]. Previous studies demonstrated that the chloroform extracts from the aerial part of C. austro-anatolica and C. cariensis had antibacterial activity against MDR Steno. maltophilia strains [17,30].
The C. pumilio EO inhibited the growth of Ent. faecalis (MIC, 125 μg/mL) more than the EO from C. helenioides did [23] and MRSA (MIC, 250 μg/mL) more than the C. baseri EO [26]. The antibacterial activity of the C. pumilio EO against MRSA may be attributed to its 15% isogermacrene D content [12]. The antimicrobial activity of the C. pumilio EO against the reference, clinical, and MDR strains might be attributed to its high content of sesquiterpenes [12]: β-caryophyllene (29.33%), isogermacrene D (17.28%), α-cyperene (14.08%), and caryophyllene oxide (10.49%). The literature reviews revealed that EOs containing high percentages of βcaryophyllene augmented antibiotic potency against Gram-negative bacteria [35]. This would suggest that C. pumilio EO is a good candidate for new formulations that can contribute to reducing AMR [35]. The current study revealed for the first time the presence of butanoic acid, 2-methyl-, 2-methylbutyl ester which has been reported for its antimicrobial activity as it is the main component in Ammi visnaga L. EO [36]. Therefore, it might also contribute to the antimicrobial activity of the C. pumilio EO. So far, the findings of the current study concerning the antimicrobial activity of the C. pumilio EO against the MDR P. aeruginosa, E. coli, and K. variicola strains can be considered as the first report among the Centaurea genus. This would underpin the great potential the C. pumilio EO has in the fight against the MDR strains with global health implications the WHO and CDC annual statistical estimates have reported [2]. As a consequence, it represents an excellent natural alternative to the inefficient synthetic anti-MDR agents.
The GC-MS analysis of the C. pumilio chloroform aerial part extract reported hydrocarbons as the most abundant constituents and showed antibacterial activity against the clinical and MDR pathogens [37][38][39][40][41]. Pentadecane and heptadecane are the major hydrocarbons and these have been reported for their antimicrobial activity [39][40][41]. Tetradecane, hexadecane, nonadecane, and heneicosane were recorded as antimicrobial agents  [41]. Eicosane, a long-chain fatty acid, has been reported for its antibacterial activity [41]. Pentadecane, tetradecane, hexadecane, nonadecane, heneicosane, eicosane, and heptacosane have previously been detected in C. napifolia, C. iconiensis, C. antiochia, C. aladaghensis, C. lanigera, C. iconiensis, and C. solstitialis, respectively [15,21,27]. The anticandidal activity of the C. pumilio chloroform aerial part extract might be accredited to 3oxo-10(14)-epoxyguai-11(13)-en-6,12-olide and curan-17-oic acid,2,16-didehydro-20-hydroxy-19-oxo,methylester which have been described as antifungal agents [41,42]. In addition, Cis-13-eicosenoic acid was detected in Camilla sinesis extract that was described for its antimicrobial activity against MRSA and MDR P. aeureginosa with MIC of 400 μg/mL and 800 μg/mL, respectively [43]. Consequently, the significant antimicrobial activity of the C. pumilio chloroform aerial part extract against the MDR P. aeureginosa and MRSA strains (MIC of 62.5 and 15.62 μg/mL, respectively) may be attributed to its content of cis-13-eicosenoic acid. The antimicrobial activities of the C. pumilio chloroform aerial part extract against the reference, clinical, and MDR pathogens are likely attributed to these bioactive compounds. The presence of these highly synergistic active compounds with high percentages in the chloroform aerial part extract may illustrate its potent antimicrobial activity against susceptible and MDR bacteria [44]. Noticeably, it is the aerial part of C. pumilio that contains more antimicrobial compounds in the EO, methanol, ethyl acetate, and chloroform extract compared to those in the root.

Conclusions
The current study is considered the first attempt to extract and assess the antimicrobial activity of the EO and extracts from C. pumilio not only against susceptible bacteria, but also MDR strains. This study does support the usage of C. pumilio as a medicinal plant as it is an extremely rich supplier of potent broad-spectrum antimicrobial bioactive compounds. The results of the research on the antimicrobial activity of C. pumilio showed that the extracts from the plant do exhibit strong activity, which is particularly promising given that antimicrobial resistance has become a major health issue worldwide. There is clear potential for C. pumilio EO and extracts to be exploited in the pharmaceutical industries and in the formulations of food additives for prophylaxis purposes. That said, further studies are mandatory to purify the chemical constituents of the antimicrobial fractions to better illustrate their mode of action. Moreover, the cytotoxicity levels of C. pumilio EO and extracts should also be studied further in vitro and in vivo to develop stable drugs for human/animal use.