Antimicrobial active herbal compounds against Acinetobacter baumannii and other pathogens

Bacterial pathogens cause a number of lethal diseases. Opportunistic bacterial pathogens grouped into ESKAPE pathogens that are linked to the high degree of morbidity, mortality and increased costs as described by Infectious Disease Society of America. Acinetobacter baumannii is one of the ESKAPE pathogens which cause respiratory infection, pneumonia and urinary tract infections. The prevalence of this pathogen increases gradually in the clinical setup where it can grow on artificial surfaces, utilize ethanol as a carbon source and resists desiccation. Carbapenems, a β-lactam, are the most commonly prescribed drugs against A. baumannii. The high level of acquired and intrinsic carbapenem resistance mechanisms acquired by these bacteria makes their eradication difficult. The pharmaceutical industry has no solution to this problem. Hence, it is an urgent requirement to find a suitable alternative to carbapenem, a commonly prescribed drug for Acinetobacter infection. In order to do this, here we have made an effort to review the active compounds of plants that have potent antibacterial activity against many bacteria including carbapenem resistant strain of A. baumannii. We have also briefly highlighted the separation and identification methods used for these active compounds. This review will help researchers involved in the screening of herbal active compounds that might act as a replacement for carbapenem.


Introduction
Acinetobacter baumannii is a Gram-negative, non-motile, and non-fermentative coccus. It is known to cause nosocomial infections such as urinary tract infections, surgical site infection, meningitis, ventilator-associated pneumonia, bacteraemia, and very rarely intra-abdominal infections and infections of skin and central nervous system (Zarrilli et al., 2009). Clinical report showed that, the hospitalized patients who are infected with A. baumannii have about 30% chance of mortality (Perez et al., 2007). Acinetobacter are usually found in the hospital environment and infect patients who are hospitalized from a long period of time with severe underlying diseases, are immunosuppressed, or subjected to invasive procedures and treated with broad-spectrum antibiotics (Perez et al., 2007). These bacteria have been reported to colonize increasingly in the respiratory tract of patients admitted in Intensive Care Unit. In case of non-intensive care unit patients high rate of colonization of Acinetobacter strains are found on skin. During outbreaks along with one or more epidemic Acinetobacter clones, there exists an endemic strain which makes it difficult to identify and control their transmission (Marchaim et al., 2007;Oteo et al., 2007).
A. baumannii have the ability to grow in diverse pH and temperature conditions and utilizes many different kinds of substrates for its growth . These are known to survive in both dry and moist condition and on inanimate objects like contaminated medical instruments including ventilators, catheters, respirometers (Cunha et al., 1980;Cefai et al., 1990;Horrevorts et al., 1995), pillows (Weernink et al., 1995), bed mattresses (Sherertz and Sullivan, 1985) etc. They are found widely in water and soil and some of them are also isolated from animals (Manchanda et al., 2010). Numerous cases of worldwide outbreaks due to A. baumannii depict increasing rates of resistance of these bacteria toward commercially available antibacterial agents.
Although no antibiotics with specific cellular targets have been isolated from plants but modified plant natural antibiotics has been more successful such as penicillin (Lewis and Ausubel, 2006). Attempts to develop potent antibiotics from plants have failed by both pharmaceutical and biotech firms. A reason for this may be the use of varying chemical strategy by the plants to control bacterial infection, in order to reduce the selective pressure for developing resistance to antibiotics. For instance, antibacterial active compounds may act quite effectively in combinations and have little potency alone. Plant alkaloid berberine has excellent antibacterial properties but it is ineffective when acting alone as it is a preferred substrate of bacterial encoded MDRs (multidrug resistance pumps). 5 ′methoxyhydnocarpin, a compound isolated from the same plant as berberine blocks the MDRs and therefore enabling berberine to act as a potent antibacterial agent in presence of this compound. So it is very necessary to first identify the antibacterial mechanism of the plants and then screen pharmaceutically to develop some effective antibiotic (Lewis and Ausubel, 2006).
Since there is no new development of antibiotics against the carbapenem resistant strains of A. baumannii therefore, it is necessary to focus on the antimicrobial activity of plant derived substances that are being used in traditional medicine worldwide (Savoia, 2012). Secondary metabolites are responsible for the antimicrobial activity of plants. In this present review, we have explained the various herbal active compounds which have potent activity against A. baumannii and other Gramnegative bacteria. This will initiate the search for new antibiotics from herbal origin against the resistant strains of Acinetobacter baumannii and this may lead to the development of new antibiotics.

Extraction of Plant Active Compounds and its Identification
Numerous works have been done to isolate and characterize bioactive compounds from plant resources that are active against Gram-positive and Gram-negative bacteria, fungi and viruses. Miyasaki reported that generally flavones, tannins and phenolic compounds are known to be active against Acinetobacter (Miyasaki et al., 2013). Many active herbal compounds have been isolated and reported for their activities. These secondary metabolites were extracted using different solvents such as methanol, ethanol, water, hydroxide, acetone, and through various techniques like HPLC, column chromatography from plant materials. After extraction of the plant materials, elucidation of the active metabolites and their structures were performed by different methods. The methods include GC-MS (gas chromatography-mass spectrometer), LC-MS (liquid chromatography-mass spectrometer), and NMR (nuclear magnetic resonance).
The efficiency for separating and identifying compounds from complex biological mixture is very high for GC-MS (Villas-Boas et al., 2005). It is capable of simultaneously identifying and profiling several compounds that possess different functions. This is mostly used for making the fingerprints of compounds that are volatile in nature which somewhat make the analyzing process repetitive and time consuming (Formisano et al., 2012;Huang et al., 2012;Wang et al., 2012). GC-MS can be used for compounds having lower molecular mass and that are volatile or semi volatile in nature (Villas-Boas et al., 2005;Dunn et al., 2011). Here, the capillary column has a low flow rate, so that output of the column can be directly put to MS ionization chamber (Oleszek and Marston, 2000;Phillipson, 2007;Daffre et al., 2008). Besides GC-MS, another most important analytical technique is LC-MS where liquid chromatography is coupled to mass spectrometry along with electrospray ionization and atmospheric pressure chemical ionization (Villas-Boas et al., 2005). It can identify and analyze a large number of compounds, present in small quantities whose molecular mass vary from 10 to 300,000 with varying chemical properties (Hagel and Facchini, 2008). As compared to GC-MS, sample preparation is quite simple in case of LC/MS (Dunn and Ellis, 2005). By LC/MS analysis, high resolution mass accuracy can be achieved which is important for analyzing herbal active compounds. This property is enhanced by the use of ultra-performance liquid chromatography. It can also be said that LC-MS is more preferred over GC-MS in reducing time and money for sample preparation. NMR spectra provide great information about the structures of the compounds along with their identification via interpretation of spectra involving chemical shift and chemical constants. It is quantitative, reproducible, non-sample destructive, and nonspecific which makes it superior to other analytical techniques (Dunn et al., 2011).
The antibacterial components of various plants, their separation and identification techniques are listed below in Table 1. Some of these herbal compounds have the ability to work alone or in presence of the other, i.e., in synergy which are also explained in Table 1.

Plant Active Compounds against Gram-Negative Bacteria
The antibacterial components of various plants were screened against a wide range of Gram-positive as well as Gram-negative bacteria mostly by MIC, disk diffusion assay and CFU. It was found that some of the compounds were more active against Gram-positive whereas some were more active against Gram-negative bacteria. The Gram-positive bacteria that are tested against different active compounds of plants include Staphylococcus aureus, Bacillus cereus, Helicobacter pylori, Salmonella enteritidis, and Clostridium jejuni (see Table 2). The Gram-negative bacteria like Klebsiella pneumoniae, Enterobacter aerogenes, Providencia stuartii, Escherichia coli, Enterobacter cloacae, and Pseudomonas aeruginosa are susceptible toward the active compounds of Hibiscus subdarifa, L. salicaria, Adansonia digitata, Commiphora molmol, etc. (see Table 2).

Plant Active Compounds against Acinetobacter baumannii
This study is aimed to review the literature regarding herbal active compounds against various bacteria, including Acinetobacter baumannii. Hence, we have listed most of the plants in Table 2, which are reported to inhibit this pathogen by varying mechanisms. L. salicaria shows significant activity against different bacteria but, especially A. baumannii and P. aeruginosa. Hence its topical form may be used to treat infections of skin and soft tissue (antiseptic), infections of burn wounds, diabetic foot and decubitus wound caused by these MDR bacterial strains. It is already known that this plant has been used as traditional medicine for many indications, but to use it clinically, several in vitro and in vivo test have to be performed (Guclu et al., 2014).
Saulnier et al. used essential oils of herbs like Syzygium aromaticum, Cinnamomum zeylanicum, and Thymus in nano medicine against multidrug-resistant A. baumannii. Cinnamaldehyde prevents the activity of amino acid decarboxylase in the bacteria (Burt, 2004), but it is unable to disorganize outer membrane of cell or deplete intracellular ATP concentration. Hydroxyl group of carvacrol and eugenol (phenolic compounds) can disrupt the bacterial cell wall. This phenomenon has the potential to decrease intracellular ATP pool and membrane potential (Ultee et al., 2002;Gill and Holley, 2006). It also results in the leakage of various substances such as ATP, amino acids, ions, and nucleic acids ultimately leading to bacterial death. MIC of active components is the same as MIC of those compounds when nano-encapsulated. Lipidic nanocapsules (LNC) can be made more effective by improving their presence in systemic circulation through the modifications on LNC surface. It can be a good alternative of antibiotics (Saulnier, 2014).
Curcumin alone had very little antibacterial activity against A. baumannii strains with high MIC (256 µg/ml). The antibacterial activity of curcumin is due to several reasons for instance, disruption of folic acid metabolism (shikimate dehydrogenase) pathway (De et al., 2009) and bacterial cell division (Rai et al., 2008). Combinatorial use of curcumin and epigallocatechin gallate (EGCG) is very much effective in increasing the inhibition level by many folds, making the MIC 4 µg/ml. This study indicates synergistic effects to prevent A. baumannii growth between curcumin and EGCG without any antagonistic effects (Betts and Wareham, 2014). In the same way epicatechin, a tea polyphenol having no antibacterial properties can potentiate theaflavin, increasing its activity against A. baumannii and S. maltophilia isolates. The probable mechanism may be that epicatechin inhibits theaflavin oxidation thus enhancing its antibacterial effect, but the exact mechanism of synergy is not yet understood and needs further study (Betts et al., 2011). Khadri, et al. reported the presence of methyl allyl trisulfide (34.61%) and diallyl disulfide (31.65%) with other compounds at relatively lower levels after GC/MS analysis in Allium sativium. It showed significant activity against P. aeruginosa in vitro and can be used for treating infections caused by this pathogen (Khadri et al., 2010). Aloe vera gel extract were reported to be more active for Gram-positive than Gram-negative bacteria. Ethanol extract was most active followed by methanol extract activity and least inhibition was exhibited by acetone extract (Lawrence et al., 2009). Magnolia dealbata extracts showed good inhibition zone of >10 mm against P. aeruginosa, Clavibacter michiganensis, A. baumannii, A. iwoffii. Generally, the active constituents of M. dealbata like honokiol and magnolol possess selective antimicrobial activities against drug resistant Gram-negative  bacterial species and fungal pathogens (Jacobo-Salcedo Mdel et al., 2011). Miyasaki et al. (2013) suggested in their studies, norwogonin (5,6,7-trihydroxyflavone) extracted from Scutellaria baicalensis, has an MIC 90 of 128 µg/ml against some strains of A. baumannii. Chebulagic acid, chebulinic acid (65% inhibition at 62.5 µg/ml), ellagic acid (67% inhibition at 250 µg/ml), corilagin, and terchebulin extracted from Terminalia chebula had lower activity against A. baumannii in vitro. Other constituents of Scutellaria baicalensis, baicalin and baicalein are also found to be active against other bacteria. Corilagin, chebulagic acid, and terchebulin of Terminalia chebula exhibits a two-step killing kinetic. The medical literature reported that many phenolic compounds of plant extracts enhance the potential of synthetic antibiotics against A. baumannii in vitro (Miyasaki et al., 2013). For instance, activity of rifampicin, coumermycin, fusidic acid, novobiocin, and chlorobiocin was enhanced by tannic acid and ellagic acid against A. baumannii in vitro (Chusri et al., 2009). Even synergy was observed between topical mafenide and green tea polyphenol against multi-drug resistance Acinetobacter baumannii in-vitro (Osterburg et al., 2009), while no effect of synergy was noted between any antibiotics for Gram-negative bacteria and norwogonin. Some Chinese medicines extracted from different plants have been reported to exhibit significant antibacterial activities. The active constituent of Rhizoma coptidis is berberine, an alkaloid possessing various antimicrobial activities. Berberine is also isolated from Berberis fremontii and Hydrastis canadensis (Lewis and Ausubel, 2006) It has anti-Herpes simplex virus effects and at moderate concentrations (30-45 µg/ml) sufficient antibacterial effect was observed along with inhibition of biofilm formation. Plant derived antibacterial molecule are generally weak but work better in synergy with antibiotics (Lewis and Ausubel, 2006). Synergism was also seen for berberine and βlactam antibiotics against multi drug resistant S. aureus. (+)-Lyoniresinol-3 alpha-O-beta-D-glucopyranoside of Cortex Lycii presented strong antimicrobial effect against multi drug resistant S. aureus isolated from patients and some pathogenic fungi, but it did not cause any haemolysis on human RBCs. This also possesses potent antifungal activities against Candida albicans. There is very limited literature concerned with the antibacterial effects of Cortex Moutan. The ethanolic extract of Cortex Moutan suppress the growth of S. aureus (Chan et al., 2011). Only paeonol is identified as active ingredients till date, which is responsible for its anti-microbial effects on C. albicans, C. tropicalis, and C. glabrata etc. (Chan et al., 2011).
A very common medicinal plant A. indica is the source of various active compounds identified by GC/MS analysis possessing versatile effects like anti-bacterial, anti-inflammatory, antioxidant activities (Nand et al., 2012). Hence further studies can be performed to see its activity against Gram-negative bacteria and especially A. baumannii.

Conclusion and Future Prospects
Emergence of resistant strains of bacterial pathogens is a major source of high morbidity, mortality, and increased cost, making its treatment much more difficult. Traditionally, plants play  Kono et al., 1997;Zhao et al., 2002;Osterburg et al., 2009;Gordon and Wareham, 2010;Betts and Wareham, 2014 A. baumannii 312-625   an important role in treatment of diseases, hence it may be used as a source to find out alternative drug to carbapenem. Plants possess secondary metabolites that are reported to be potentially active against a wide variety of bacteria. It was also found that the herbal compounds are weak antibacterials but showed better antimicrobial activity when used in synergy with other antibiotics. The advancement in the techniques for separation, purification and identification of bioactive compounds make it possible to chemically and structurally identify these compounds. In present review, we have made an effort to list a number of plant active compounds that may have potent antibacterial activity against some Gram-positive and Gram-negative bacteria including carbapenem resistant strain of A. baumannii. Hence, the use of different plant natural compounds as antibacterial agents is an interesting strategy for discovering bioactive products that could become useful therapeutic tools. This review will help researchers involved in the screening of herbal active compounds that might act as a replacement for carbapenem, a β-lactam. In this review we found a compound named norwogonin which has good antibacterial activity (MIC 90 -128 µg/ml) against A. baumannii. Hence, special emphasis can be given on this compound in further studies in order to use it as an alternative of carbapenem. Effect of this compound on other bacteria can also be screened. There are some plants shown in Table 2 that shows good activity against many bacteria like Allium sativum, Adansonia digitata, Hibiscus subdarifa, etc., have not been tested against A. baumannii. Therefore, activities of these plants can also be tested against A. baumannii. Screened herbal active compound may be tested in the future for its antimicrobial activity. In-silico approach can also be used to modify the structure of active compound for its better antibacterial activity against carbapenem resistant strain of A. baumannii. Potential plant active compound may be chemically synthesized and used as an alternative to carbapenem.