The bioactivity of plant extracts against representative bacterial pathogens of the lower respiratory tract

Background Lower respiratory tract infections are a major cause of illness and death. Such infections are common in intensive care units (ICU) and their lethality persists despite advances in diagnosis, treatment and prevention. In Mexico, some plants are used in traditional medicine to treat respiratory diseases or ailments such as cough, bronchitis, tuberculosis and other infections. Medical knowledge derived from traditional societies has motivated searches for new bioactive molecules derived from plants that show potent activity against bacterial pathogens. Therefore, the aim of this study was to evaluate the effect of hexanic, chloroformic (CLO), methanolic (MET) and aqueous extracts from various plants used in Mexican traditional medicine on various microorganisms associated with respiratory disease. Methods thirty-five extracts prepared from nine plants used in Mexican traditional medicine for the treatment of respiratory infections were evaluated against 15 control bacterial species and clinical isolates. Results Both chloroformic (CLO) and methanolic (MET) extracts of Larrea tridentata were active against Methicillin-resistant S. aureus, B. subtilis and L. monocytogenes. A MET extract of L. tridentata was also active against S. aureus, S. pneumoniae, S. maltophilia, E. faecalis and H. influenzae and the CLO extract was active against A. baumannii. An Aqueous extract of M. acumitata and a MET extract of N. officinale were active against S. pneumoniae. CLO and MET extracts of L. tridentata were active against clinical isolates of S. aureus, S. pneumoniae and E. faecalis. Conclusion Overall, our results support the potential use of L. tridentata as a source of antibacterial compounds.

Infections with these bacteria are associated with high morbidity and mortality, especially in immunocompromised patients [7][8][9][10]. Methicillin-resistant S. aureus (MRSA) is of particular concern in the United States since approximately 60% of all staphylococcal infections in the ICU are caused by MRSA [11,12]. Penicillin-resistant S. pneumoniae and vancomycin-resistant enterococci are an additional cause for concern in the clinical setting [13].
In Mexico, some plants are used in traditional medicine to treat respiratory diseases or ailments such as cough, bronchitis, tuberculosis and other infections [14,15]. The known success of these traditional therapies has guided the search for new chemotherapeutic alternatives to fight respiratory and other infections caused by drug-resistant bacteria [16][17][18]. Therefore, the aim of this study was to evaluate the effect of hexanic, chloroformic (CLO), methanolic (MET) and aqueous extracts from various plants used in Mexican traditional medicine on various microorganisms associated with respiratory disease.

Plant material and preparation of extracts
We reviewed Mexican ethnobotanical literature and selected a group of plants used by traditional healers to treat respiratory infections [14,15] Approximately 50 g of dried and ground plant material was successively extracted by maceration with hexane, chloroform, methanol and water. Organic extracts were concentrated in vacuo to dryness, and aqueous extracts were freeze-dried. All extracts were stored at 4°C until use.
Hexanic, CLO, MET and aqueous extracts were made from the following: C. aurantifolia, C. sinensis, F. vulgare, L. tridentata, N. officinale, O. europaea, M. pulegium, R. centifolia. Only CLO, MET and aqueous extracts were prepared form M. acuminata because of limited plant material availability.
Stock solutions were prepared from extracts at a concentration of 20 mg/ml. For organic extracts, DMSO was used as a solvent. Aqueous extracts were prepared in distilled water and sterilized by filtration through a 0.22 μm membrane. Before testing, strains were inoculated onto plates prepared with 5% blood agar and cultured overnight at 37°C. For H. influenzae, strains were inoculated onto chocolate agar and incubated in a 5% CO 2 atmosphere for 48 h. Turbidity was adjusted to 0.5 of the McFarland standard to prepare the test inoculum. Afterwards, 10 μl were transferred into 11 ml Mueller Hinton broth to achieve 5 × 10 5 CFU/ml. HTM medium was used for H. influenzae and Mueller Hinton with TES/lysed horse blood was used for S. pneumoniae.

Screening of antimicrobial activity and reference drugs
Minimal inhibitory concentration (MIC) for each extract was determined for both Gram-negative and Gram-positive bacteria by the microdilution method [19] with dilutions ranging from 250 to 7.8 μg/ml. Extracts that caused complete inhibition of growth at 24 h were considered active and used for the subsequent assays. All assays were performed in duplicate.
Two extracts with the broadest spectrum of activity were further tested against clinical isolates of susceptible species collected at the Hospital Universitario "Dr. José Eleuterio" Gonzalez and deposited at the Microbiology Department, School of Medicine, UANL. These isolates included A. baumannii (n = 25), E. faecalis (n = 15), S. aureus (n = 25), and S. pneumoniae (n = 13; total n = 78). H. influenzae was not included because clinical isolates were not available.
For all assays, adequate media without drug or extract was used as control.
As a reference for the screening assays, MICs for the antimicrobial drugs ceftazidime (Caz), ciprofloxacin, levofloxacin (Lv) and vancomycin (Va) were determined by the broth microdilution method recommended by the Clinical and Laboratory Standards Institute [20]. As a reference for the assays with clinical isolates, we evaluated the susceptibility pattern for each clinical isolate against the following commonly used antibiotics: Caz, Lv, Va, minocycline, penicillin, piperacillin tazobactam, amoxicillin clavulanic acid, ampicillin, linezolid, meropenem, amikacin, cefepime, and/or ceftriaxone.

Discussion and conclusion
Among the plant extracts that showed antibacterial activity, the CLO and MET extracts of L. tridentata showed the broadest and most promising spectrum of activity. In order to confirm this activity, we tested those extracts against clinical isolates of species that showed sensitivity to these extracts in the screening assay.
Both extracts confirmed the antibacterial activity, not only in control strains, but also against strains isolated from patient's clinical samples. The best activity was observed against S. pneumoniae (MICs 80 = 31.25 μg/ml). When tested against S. pneumoniae, the antimicrobial activity of CLO and MET extracts of L. tridentata were comparable to the activity observed for reference antimicrobial drugs. It is possible that further isolation of the active compound(s) will render lower MIC values.
Both the CLO and MET extracts of L. tridentata also showed good activity against E. faecalis (MICs 50 = 250 μg/ ml) and S. aureus (MICs 80 = 125 μg/ml). These results are quite interesting, given the high drug-resistance observed for those clinical isolates. The CLO and MET extracts of L. tridentata were active against MDR clones of S. aureus. These results are particularly important because of the increase in morbidity and mortality related to clones of MDR S. aureus in the last two decades [11].
No activity was detected against any clinical isolates of A. baumannii. Nevertheless, this is not a surprising result sinceclinical isolates of A. baumannii show a strong drugresistance, so there are likely multiple antimicrobialresistance mechanisms in these strains.
The CLO and MET extracts of L. tridentata showed different activity spectra. Verastegui et al. reported that ethanolic extract of L. tridentata was active against L. monocytogenes, Clostridium perfringens, Shigella dysenteriae, Yersinia enterocolitica and Proteus vulgaris, with a MIC ranging from 10 to 19 μg/ml, yet they did not find activity against E. coli [21]. We found that CLO and MET extracts of L. tridentata showed activity against L. monocytogenes and no activity against E. coli. This confirms the antibiotic activity of L. tridentata extracts; however, it is difficult to compare our results directly with Verastegui et al. because we did not include these ethanolic extracts in our study.
L. tridentata (Creosote bush) is an abundant plant of Mexican and US-American deserts. The potent antioxidant nordihydroguaiaretic acid (NDGA) has been identified from this plant [22]. This lignan is a potential therapeutic molecule for Sjorgren-Larsson syndrome, cardiopathies, skin cancer prevention, and viral infections [23]. We presume that the compounds present in our L. tridentata CLO extract are not NDGA because they have a different polarity. Thus, they are potentially novel antibacterial molecules. Antimicrobial properties for some plant species tested in the study have previously been described but not with a special focus on respiratory tract pathogens and the inclusion of some bacterial MDR clones.
Our results showed that the MET extract of N. officinale and the aqueous extract of M. acuminata were active against S. pneumoniae. N. officinale has been used for the treatment of renal, liver disease [24], and has also been shown to reduce oxidative stress in hypercholesterolemic rats [25]. This plant has also been shown to have cardioprotective activity [26] and antimycobacterial potential [27]. Indeed, several antimicrobial compounds have been identified in N. officinale. Specifically, a phenyl-phenalenone has been isolated and evaluated for leishmanicidal activity and isolated phytoalexins are known to have antifungal activity [28]. We found that the methanolic extract of N. officinale is active against S. pneumoniae.
Our results also demonstrated the activity of M. acuminata aqueous extract against S. pneumoniae ATCC 49619 (250 μg/ml). Similarly, activity of M. acuminata MET extract against Mycobacterium tuberculosis has been previously reported [27]. To our knowledge, there is no other antimicrobial activity report for extracts form M acuminata.
Our findings do conflict with some of the previously published results on the same plant extracts. We did not find antibacterial activity in several plants whose antimicrobial effects have been documented. For example, we did not find any antibacterial activity in extract from O. europaea, yet another group isolated the aldehyde hexanal extract from O. europaea and reported bioactivity against multiple types of microorganisms [29]. In addition, a terpenoid extracted from C. sinensis with antifungal activity has been isolated from the fruit peel [30], yet we found no effect of the extract in our study.
The differences between our study and theirs could have multiple explanations. The strains or clones and the testing concentrations differed and perhaps local environmental factors that affect the potency of medicinal plants, such as temperature, rainfall, day length and soil characteristics may have differed between the plant samples used for each study. Also, it is important to consider differences in the conditions of extract preparation and in the extract concentrations tested.
Our study has provided and confirmed previous evidence that L. tridentata is a promising alternative source for antimicrobial compounds. It is particularly valuable in the context of the bacterial resistance that is prevalent. Our group is currently conducting a study to isolate the active compounds in this extract.
Lastly, the activity observed for L. tridentata provides a rationale for its use in the treatment of respiratory infectious diseases in traditional medicine.