Antimicrobial activity of Luma apiculata against resistant strains of Staphylococcus aureus and Klebsiella pneumoniae

Bacterial resistance is the mechanism through which the bacteria can decrease the action of antimicrobial agents. Studies have reported an antimicrobial action of Luma apiculata, however, there are no in vitro studies demonstrating the effect of this plant against multidrug-resistant bacteria. The objective of this study was to explore the antimicrobial properties of alcoholic extract of Luma apiculata against standard multi-resistant bacterial pathogens. Multidrug-resistant strains of Staphylococcus aureus ATCC 43300 (MRSA) and Klebsiella pneumoniae ATCC BAA 1705 (CRKP) were incubated independently for 72 h at 37oC with different concentrations (1-10,000 μg/mL) of alcoholic extract of Luma apiculata. The growth curve was studied up to 72 hours. The minimum inhibitory concentration (MIC) was determined by broth macrodilution method. The agar diffusion method was used to examine bacteriostatic activity and bactericidal activity was evaluated by a commercial kit and analyzed by flow cytometry. Analyses of chromatographic data, as well as mass spectrometry, were performed to determine the main compounds present in the extracts. Luma apiculata leaves extracts showed bactericidal activity and was effective attenuating growth in both bacterial strains. Thus, Luma apiculata could represent a natural alternative against multidrug-resistant bacterial strains. However, further studies should be undertaken to test their clinical utility.


Introduction
Antimicrobial resistance (AR) represents an important health issue that requires worldwide attention. The importance of AR has grown gradually becoming a global issue mainly because it can be transmitted between different hosts and rapidly spreads between continents [1,2]. In recent years, antibioticresistant pathogens from the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter spp.) have been frequently detected in patients, representing an emerging health threat [3]. The emergence of antibiotic-resistant forms of pathogenic S. aureus is a worldwide problem in clinical medicine [4]. Furthermore, methicillin-resistant Staphylococcus aureus (MRSA) remains as a major global cause of health-care-associated infections [5]. Klebsiella pneumoniae is an opportunistic pathogen in immunocompromised individuals that causes a wide range of infections, including pneumonia, urinary tract infection, bacteremia, and meningitis [6]. In the last two decades, strains confirmed to produce Klebsiella pneumoniae carbapenemase (KPC) have been attributed to numerous nosocomial infections worldwide. Despite the fact that Klebsiella pneumoniae is the main producer of -lactamase type KPC, other species such as Enterobacteriaceae (Klebsiella oxytoca, Enterobacter spp., Escherichia coli, Salmonella spp., Citrobacter freundii, and Serratia spp.) and Pseudomonas aeruginosa also produce enzymes that confer resistance to -lactamic agents [7,8].
In recent years, there has been an increased interest in studying the antibacterial properties of natural sources such as plants. Antimicrobial compounds from these natural sources, including essential oils or plant extracts, are currently available for their use in alternative therapies [9]. A number of studies from different plant species have shown antibacterial properties [10][11][12]. The arrayan (Luma apiculata (DC.) Burret) is a Chilean myrtle that has been used in traditional medicine [13] . The medicinal properties of Myrtle leaves include aromatic, astringent, balsamic and anti-inflammatory actions [14]. Previous reports have shown an inhibitory activity of the xanthine oxidase enzyme by Paraguayan and Chilean Myrtaceae species commonly used in folk medicine to treat gout [15] and also as astringent [16]. Leaves of Luma apiculata are used to treat diarrhea, dysentery, and ingestion [17]. In addition, Simirgiotis et al. [18] described the phenolic compounds pattern, highlighting its high polyphenolic contents and demonstrated the high antioxidant capacity in leaves of Luma apiculate. Despite the traditional use of Luma apiculata, few investigations have been performed to prove its therapeutic effect as anti-infective agent to establish potential medicinal activity against persistent infections. The objective of this study was to explore the antimicrobial properties of alcoholic extracts of Luma apiculata leaves against standard multiresistant bacterial pathogens.

Proof of resistance and sensitivity to antibiotics
Although ATCC has tested every one of the strains to verify species, KPC production, and multidrug resistance, we confirmed the relative resistance of MRSA and CRKP strains using disk diffusion method with cefoxitin (30 µg) and meropenem (10 µg), and sensitivity with vancomycin and gentamicin (10 µg), respectively ( Figure 1). The agar plates were incubated at 37 ºC for 24 h. Effect of plant extract on bacterial growth curve To evaluate the effect of plant extracts on bacterial growth curve, different concentrations of extract were prepared (1, 10, 100, 1000 and 10000 µg/mL). All concentrations of both bacterial strains were incubated at an initial concentration of 0.5 McFarland (1.5 x 10 8 cells per cm 3 ). Bacterial concentrations and bacterial growth rates were calculated by measuring the optical density (OD) at the wavelengths of 600 nm at 0, 3, 6, 24, 48 and 72 h (OD of 0.1 corresponds to a concentration of 1.5 x 10 8 cells per cm 3 ). Bacterial strains without plant extracts were used as controls.

Determination of minimum inhibitory concentration (MIC) of plant extracts
For the determination of MIC, the broth macrodilution susceptibility assay was performed, according to standard M100-S9 of the National Committee for Clinical Laboratory Standards [19]. Six dilutions series of plant extracts were prepared from 1 to 10,000 µg/mL, into test tubes containing 10 mL Mueller Hinton broth medium. The bacterial broth cultures were adjusted to 0.5 McFarland turbidity standards and added into each of the test tubes. The medium containing a mixture of bacteria and extracts was incubated at 37 ºC for 24 h in a shaker water bath. Bacterial strains without plant extracts were used as controls.

Antimicrobial susceptibility testing
The antibacterial activity assay of plant extracts was developed using the agar diffusion method. The MRSA and CRKP strains were performed by Kirby-Bauer method on Muller Hinton agar plates. Whatman filter No.1 paper discs were soaked with 10 L of extract in concentrations of 1, 10, 100, 1000 and 10,000 µg/mL and air dried before the assay [20]. Cultures were incubated at 37 ºC for 24 h prior to testing, using the criteria of standard zone sizes of inhibition according to Clinical and Laboratory Standards Institute (CLSI) guidelines [19]. After the period of incubation, the antimicrobial activity was measured in millimeters of the zone of inhibition and categorized as sensitive or resistant.

Bacteriostatic activity of plant extracts
To estimate the bacteriostatic activity of alcoholic extracts of L. apiculata, each Mueller Hinton broth tubes used to calculate the MIC was transferred onto the surface of Muller Hinton agar plates and incubated at 37 ºC for 24 h. Inhibition of bacterial growth was considered as bacteriostatic activity of that extract.

Bactericidal activity of plant extracts
To assess the effect of plant extracts on bactericidal activity, concentrations of 100, 1,000 and 10,000 µg/mL of each extract were prepared. Bacterial strains without plant extracts were used as controls. The bacterial strains were incubated at an initial concentration of 0.5 McFarland (1.5 x 10 8 cells per cm 3 ) at 37 ºC for 72 h. Direct measurement of bactericidal activity was assessed by using LIVE/DEAD ® BacLight bacterial viability Kit (Molecular Probes, Eugene, OR, USA). This assay uses twocolor fluorescences, which differs in the ability to penetrate cell membranes. The green fluorescent SYTO ® 9 stains the nucleic acids of both living and dead bacteria, while Propidium Iodide (red) labels the nucleus of bacteria with damaged and leaky membranes. The fluorescence analysis was performed using a FACSCanto TM II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were acquired and analysed with the software FACSDiva version 6.1.3 (Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA). At least 10,000 events were acquired for each sample. Fluorochrome excitation was carried out using a 488 nm argon-ion laser. Green fluorescence was collected with a 530/30 nm bandpass filter and orange fluorescence through a 585/42 nm bandpass filter, both were analyzed on logarithmic scales. The analyses were performed using the Turbo V (R) ion spray in positive and negative ionization mode by using the information dependent acquisition (IDA) method editor. Enhanced MS (EMS) spectra were acquired between m/z 100-1,000 at scan speed of 1,000 Da/s. Enhanced production ion (EPI) spectra were acquired between m/z 50-1,000 at scan speed of 4,000 Da/s which provided a fragment footprint of compounds of interest.

Statistical analysis
All trials were repeated at least five times. The experiments were carried out in duplicate and results were expressed as mean ± S.D. Statistical evaluation was performed with the Prism 6 software package (GraphPad, La Jolla, CA, USA), applying D`Agostino test to assess Gaussian distribution. Analysis of variance (ANOVA) was applied to determine the effect produced by different concentrations of the plant extracts and Bonferroni post hoc test was used to perform multiple comparisons. Statistical significance was established for P < 0.05.

Effect of plant extracts on bacterial growth curve
Description of the bacterial growth curves is shown in (Figure 2). Exponential growth phase and stationary phase were observed early at 24 h, and 72 h, for S. aureus and K. pneumoniae respectively. The bacterial growth of S. aureus and K. pneumoniae was diminished 6-fold by the addition of alcoholic extract of L. apiculata, compared to the profiles without any plant extract (P < 0.0001). L. apiculata did not alter the growth curve of S. aureus between concentrations of 1 µg/mL to 100 µg/mL dry material, compared with the control group (P > 0.05). Contrary, we observed that growth curves of S. aureus were reduced significantly with concentrations of 1,000 µg/mL and 10,000 µg/mL dry material after 6 h and 48 h of incubation respectively (P < 0.001), compared with the control group (Figure 2A). Treatment with an alcoholic extract of L. apiculata against K. pneumoniae revealed no significant difference among dry material concentrations of 1 µg/mL to 100 µg/mL compared to the untreated control (P > 0.05). However, significant differences were found after 6 h with 10,000 µg/mL and after 24 h with 1,000 µg/mL of dry material compared to the untreated control (P < 0.001; Figure  2B).

Determination of minimum inhibitory concentrations
Determination of MIC was not possible due to the appearance of intense turbidity during the test conditions for all plant extracts. The same growth inhibitory activity can be observed with the agar diffusion method, which is adequate to assess the possible antimicrobial activity of natural extracts [21]. Sensitivity analysis to extracts by the agar diffusion method A clear circular area of inhibition was obtained from the agar diffusion test. The bacterial growth inhibition for S. aureus using 30 µg cefoxitin disc ( Figure 3F) as resistant control, was approximately 16 mm (specific breakpoint ≤21 mm for resistant and ≥22 mm for susceptible) and using vancomycin-MIC ( Figure 3G) as sensitive control, it was 0.75 μg/mL (specific breakpoint ≤ 2 µg/mL sensitive). When we evaluated growth inhibitory activity of plant extract against S. aureus, only concentrations of 1,000 μg/mL and 10,000 μg/mL showed a significative inhibitory activity ( Figures 3A,  B, C respectively). At the concentration of 1,000 μg/mL an inhibition zone of 9 mm was observed ( Figure 3D). The maximum inhibition zone (14 mm) was detected at the concentration of 10,000 μg/mL ( Figure 3E). The bacterial growth inhibition for K. pneumoniae using 10 µg meropenem disc ( Figure 4F) as resistant control, was approximately 15 mm (specific breakpoint ≤19 mm for resistant and ≥22 mm for susceptible) and using 10 µg gentamicin disc ( Figure 4G) as sensitive control, it was approximately 21 mm (specific breakpoint ≥15 mm sensitive). Discs with concentrations of 1, 10 and 100 µg/mL were found to be resistant, showing no signs of inhibition ( Figure 4A, B, C respectively). At the higher concentrations of 1,000 and 10,000 μg/mL a small inhibition zone (7 mm) was observed ( Figures 4D and E, respectively).

Bacteriostatic activity of plant extracts
Bacteriostatic activity of L. apiculata was only observed against MRSA. When concentrations of 10,000 µg/mL were challenged against MRSA an inhibition of the development of bacterial colonies was observed ( Figure 5F).

Bactericidal activity of plant extract
The bactericidal activity of plant extracts against S. aureus ATCC 43300 ( Figure 6A) and K. pneumoniae ATCC BAA 1705 ( Figure 6B) was statistically significant at the concentrations of 1,000 and 10,000 µg/mL compared to control. The bactericidal activity detected in the alcoholic extract of L. apiculata against S. aureus ATCC 43300 was significantly more effective than for K. pneumoniae ATCC BAA 1705 at the concentration of 1,000 and 10,000 μg/mL.

Discussion
Antimicrobial resistance has turned into a severe health threat concern worldwide promoting scientific interventions and encourages to find new therapeutic alternatives. In March of 2015, 36 novel broad-spectrum antimicrobials that can treat serious bacterial infections are under clinical trials [22]. Plant species have recently received attention for their antimicrobial compounds usually stored as secondary metabolites [11]. In our study, an alcoholic extract from leaves of Luma apiculata was used. The alcoholic extraction is more effective because the concentration required to inhibit the bacteria growth is lower, and the bacterial inhibitory effect is stronger than the aqueous extract [23]. Although flavonoids are soluble in water, better recovery is achieved by using polar solvents such as ethanol, obtaining a greater amount of phenolic and flavonoid compounds which are attributed with antibacterial activity [24]. Luma apiculata showed antimicrobial activity only with the highest concentrations used in this study. We did not test for cytotoxicity at these concentrations but there are no reports of cytotoxicity of these concentrations in somatic cells. In terms of selectivity, a number of studies have shown that non-cancer cells are less sensitive to plant extracts than tumor cell lines, which can be explained by the growth rate of tumor cells compared with non-cancer cells [25]. In addition, a toxicity study suggested that myricetin, a compound present in L. apiculata extract, does not exert cytotoxicity towards normal breast epithelial MCF10A cells, hence, researchers concluded that the compound is safe for further in vivo studies [26]. This strongly suggests that selective toxicity of L. apiculata is an important factor that should be investigated. The results of this study show that plant extract inhibited bacterial growth with the highest concentrations used. The inhibitory action of bacterial growth can be due to the presence of monoterpenes in the plant [27]. These organic compounds exert a protective function against biotic attacks such as infections caused by viral, bacterial and fungal pathogens. Possible mechanisms of action may be due to the lipid bilayer disruption by the terpenes [12, 28].

Table 1. Identification of phenolic compounds in Luma apiculata leaves by an HPLC/UV-ESI MS MS system. The analyses were performed using the Turbo V(R) ion spray in positive (A) and negative (B) ionization mode A B
The bactericidal activity detected in the alcoholic extract of L. apiculata against S. aureus was significantly more effective than for K. pneumoniae. This is probably due to the composition of the outer membrane of both bacteria. According to Pitout and Laupland [29] Gram-negative bacteria are more resistant to the extracts than Grampositive bacteria, because they have hydrophilic polysaccharide chains, which act as a barrier against extracts or essential oils.
Several studies suggest that the mechanism of action of some antimicrobial compounds, such as phenolic compounds, involves damage to both, wall and bacterial cell membrane, which facilitates the leaking of intracellular constituents like proteins, inorganic ions and several enzymes from the cells, triggering a permanent cell damage [30]. More importantly, it has also been reported antimicrobial activity; for example, flavonoids have been found to be effective against Gram-positive bacteria [36]. Another study revealed bacteriostatic activity in several flavonoids tested separately, confirming that quercetin was the most effective inhibitor [37]. In addition, when flavonoids are used in combination with antibiotics show synergistic effects against strain bacterial [30]. This study may not be adequate to suggest a potential new antibiotic agent, considering that antibacterial activity was observed at higher concentrations than those reported in other plant extracts. However, this approach should be considered as a preliminary step to find out new promising molecules with antibacterial activity and to unravel their possible mechanism of microorganism inhibition. Further studies are still needed to assess the influence of different portions of the plant extracts including flowers, leaves or stems, on the bacterial growth rate and as well the evaluation of possible synergistic effects with antibiotics or other medicinal plant extracts.

Conclusion
Luma apiculata has antibacterial activity against standard multidrug-resistance strains of S. aureus and K. pneumoniae. The bactericidal activity detected in the alcoholic extract of L. apiculata against S. aureus was more effective than for K. pneumoniae. Flavonoids were the principal compounds detected in the ethanolic extract of L. apiculata.