Phytochemistry and Preliminary Assessment of the Antibacterial Activity of Chloroform Extract of Amburana cearensis (Allemão) A.C. Sm. against Klebsiella pneumoniae Carbapenemase-Producing Strains

The chloroform extract of the stem bark of Amburana cearensis was chemically characterized and tested for antibacterial activity.The extract was analyzed by gas chromatography and mass spectrometry. The main compounds identified were 4-methoxy-3-methylphenol (76.7%), triciclene (3.9%), α-pinene (1.0%), β-pinene (2.2%), and 4-hydroxybenzoic acid (3.1%). Preliminary antibacterial tests were carried out against species of distinct morphophysiological characteristics: Escherichia coli, Salmonella enterica Serotype Typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus. The minimum inhibitory concentration (MIC) was determinate in 96-well microplates for the chloroform extract and an analogue of themain compound identified, which was purchased commercially.We have shown that plant's extract was only inhibitory (but not bactericidal) at the maximum concentration of 6900 μg/mL against Pseudomonas aeruginosa and Bacillus cereus. Conversely, the analogue 2-methoxy-4-methylphenol produced MICs ranging from215 to 431 μg/mL against all bacterial species.New antibacterial assays conducted with such chemical compound against Klebsiella pneumoniae carbapenemase-producing strains have shown similarMICresults and minimumbactericidal concentration (MBC) of 431 μg/mL.We conclude that A. cearensis is a good source of methoxy-methylphenol compounds,which could be screened for antibacterial activity againstmultiresistant bacteria fromdifferent species

However, although A. cearensis has been broadly used in treatment of respiratory infections, only a few studies report antimicrobial activity in plants' extracts [9,10], and, therefore, its potential as a source of new antimicrobials was not fully investigated.
The spread of multidrug resistance among bacteria from various sources [11][12][13][14][15][16] has made natural product researchers increase their effort on screening plant extracts for compounds with broad spectrum of antimicrobial activity. For example, Tragia involucrata L., Citrus acida (Roxb. Hook.f.), and Aegle marmelos (L.) Correa ex Roxb. showed wide inhibitory action against several multidrug-resistant human pathogens, particularly, Burkholderia pseudomallei and Staphylococcus aureus, which was related to the high contents of phenolic or polyphenolic compounds in methanol extracts [17,18]. Likewise, recent antibacterial assays with stem bark ethanol extracts of A. cearensis have shown growth inhibition of a broad range of pathogens of veterinary interest [10]. In particular, hospital-acquired infections caused by Klebsiella pneumoniae (Enterobacteriaceae) have increased in recent years due to emergence of carbapenemase-producing strains [19,20]. These bacteria are capable of hydrolyzing carbapenems, penicillins, cephalosporins, and aztreonam [21], and, therefore, the search for new therapeutics is forthcoming.
Here, a preliminary assessment of the antibacterial activity of A. cearensis chloroform extracts against human clinical isolates of K. pneumoniae carbapenemase-producing strains was conducted. Moreover, the plants' extract was chemically characterized and an analogue of the major compound identified was also tested against bacteria. Although the chemical composition of plant extracts varies under influence of seasonal and climatic conditions [22], in the present study, we report A. cearensis as a new source of methoxymethylphenol compounds with antibacterial activity. These are phenol derivatives with potential applications as antiseptics and biocides. These data are discussed in light of previous studies.

Plant Extract.
Fresh plant material was collected and dried in oven at a temperature range of 45 ∘ C to 50 ∘ C for 48 h. The dried material was ground in a blade mill to obtain a fine homogeneous powder. This material was weighed and extracted by maceration using chloroform (P.A.) as solvent extractor in the ratio of 1 : 3 (w : v). The resulting mixture remained for 48 hours under agitation every two hours. The extract was filtered and concentrated in a rotary evaporator under reduced pressure at a temperature of 45 ∘ C for complete solvent removal. Stock solutions were prepared with extracts using dimethyl sulfoxide (DMSO) as solvent (100 mg/mL), which were kept in a refrigerator at −20 ∘ C until use [23].

Analysis by Gas Chromatography Coupled to Mass Spectrometry (GS/MS).
The plant extract was analyzed by GS/MS using a Varian 431-GC chromatograph coupled to a Varian 220-MS mass spectrometer, equipped with a J & W Scientific DB5 fused silica capillary column (30 m × 0.25 mm × 0.25 mm). The temperature of the injector and detector was set at 260 ∘ C with the furnace temperature programmed in a range of 60-240 ∘ C at 3 ∘ C/min. The mass spectra were obtained with a 70 eV electron impact, 0.84 scan/sec m/z 40-550. The carrier gas used was helium at a flow rate of 1 mL/min. A stock solution of 2 mg/mL was prepared and 1.0 L was injected for analyses. The identification of the constituents was carried out by comparison with previously reported values of retention indices, obtained by coinjection of oil samples and C 11 -C 24 linear hydrocarbons and calculated using the van Den Dool and Kratz equation [24], by direct comparison of the spectra with spectra stored in libraries of equipment (NIST21 and NIST107) as well as with the spectra and retention times of authentic compounds reported previously in the literature for comparison. Subsequently, the MS acquired for each component was matched with those stored in the NIST21, NIST107 mass spectral library of the GC-MS system and with other published mass spectral data [25].

Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
The MIC of the plant extract was determined by the microdilution method [34] in 96-well plates at concentrations ranging from 6.7 to 6900 g/mL. In addition, the analogue 2-methoxy-4-methylphenol of the main compound identified in plant extracts (4-methoxy-3-methylphenol) was purchased commercially (Sigma) and also investigated. The density of the bacterial suspension was adjusted to approximately 10 5 CFU/mL, and the results were read after an incubation period of 24 hours at 37 ∘ C. The MIC was the lowest concentration causing inhibition of visible growth. In this case, aliquots of 0.1 mL were transferred to plates containing Mueller-Hinton agar. The minimum bactericidal concentration (MBC) was considered the lowest concentration that resulted in no growth after incubation for 24 h at 37 ∘ C. All assays were performed in duplicate.

Results and Discussion
The Brazilian floras are worldwide known as a source of biologically active compounds with biodegradable properties, and, therefore, screening of plant extracts is often a first step procedure for isolation of phytochemicals [23]. However, studies that correlate antibacterial activity of extracts to the presence of specific compounds are rare. For instance, ethanol extracts of the stem bark of A. cearensis were reported to be inhibitory against Staphylococcus epidermidis, S. aureus, Klebsiella spp., Salmonella spp., Enterobacter aerogenes, Streptococcus pyogenes, Proteus mirabilis, Pseudomonas aeruginosa, and Shigella flexneri, but no chemical characterization was performed [10]. Here, we report the phytochemistry and preliminary assessment of A. cearensis chloroform extracts 4 Evidence-Based Complementary and Alternative Medicine  Not reported [32] Trunk bark Ethanol Coumarin and vanillic acid Not reported [33] as a source of novel antimicrobials against multiresistant Klebsiella strains. The GC/MS data showed a major peak at the retention time of 7.8 min with relative concentration of 76.7% (Figure 1). The interpretation of the mass spectra in comparison with spectra reported in the literature led to identification of the benzenoid 4-methoxy-3-methylphenol. Other compounds included tricyclene (3.9%), -pinene (1.0%), -pinene (2.2%), and 4-hydroxybenzoic acid (3.1% anthraquinone (chrysophanol), with predominance of coumarins being often reported [35]. The initial screening for antibacterial activity has shown that plants' chloroform extract was only inhibitory against P. aeruginosa and B. cereus at the highest concentration of 6900 g/mL ( Table 2). P. aeruginosa is responsible for different etiological processes in immunocompetent and immunocompromised patients in hospitals, whereas B. cereus is an opportunistic pathogen [36,37]. On the other hand, the analogue compound 2-methoxy-4-methylphenol showed broad spectrum activity against all bacteria tested (Table 3). Indeed, the MIC ranged from 215 to 431 g/mL against K. pneumoniae carbapenemase-producing strains being also bactericidal at 431 g/mL ( Table 3). The carbapenems imipenem, meropenem, and ertapenem are often used as last therapeutic choices against gram-negative multidrug-resistant bacteria [38]. Moreover, these bacteria usually show resistance to aminoglycosides and fluoroquinolones due to the presence of gene gnr and kpc [39]. Thus, the risk of nosocomial infections is higher for patients in hospital's intensive care units [40,41].
According to PubChem Compound Database, 4methoxy-3-methylphenol is also known as 14786-82-4, AG-D-93185, NSC168522, AC1L6RKC, SureCN263095, and 4-methoxy-3-methylphenol [42]. Although anticancer assays were previously carried out with the compound, it was reported to be inactive against tumor model L1210 leukemia in mice [42]. While we were not aware of previous antimicrobial studies conducted with 4-methoxy-3-methylphenol, we hypothesize that the action mechanism of the molecule against bacteria is possibly due to 3methylphenol compound (m-cresol ) well known as an oxidizing agent [43]. Yet, m-cresol is a methyl derivative of phenol that has been used as precursor of amylmetacresol present in commercial antiseptic formulation.
The array of chemical compounds in A. cearensis extracts varies despite the type of solvent used (Table 4). Accordingly, we have shown that chloroform extracts of A. cearensis are good sources of methoxy-methylphenol compounds with antibacterial activity against several bacterial species and clinical isolates of multidrug-resistant K. pneumonia. Thus, the plants' chloroform extract could be exploited as a source of antiseptics or biocides against drug-resistant bacteria from different species.