Antibacterial activity of Brazilian Northeast plants against Corynebacterium pseudotuberculosis

The production of small ruminants is an important economic activity of the brazilian Northeast, but some diseases have a high prevalence in this region, such as caseous lymphadenitis (CL), caused by Corynebacterium pseudotuberculosis. The treatment of CL is often ineffective, which justifies the search of new active principles from plants, mainly of the region, to have an accessible treatment. For this reason, the present study was carried out to evaluate the in vitro antibacterial activity of Annona squamosa, Azadirachta indica, Allium sativum, Prosopis juliflora and Portulaca oleracea against C. pseudotuberculosis. Agar well diffusion assay (AWD) and broth microdilution to determine minimum inhibitory concentration (MIC) and bactericidal concentration (MBC) evaluated the antimicrobial activity. The highest antibacterial potential was obtained by ethanolic extracts of A. indica leaf (MIC 0,12 mg/mL for 2 strains), A. squamosa stalk (MIC 0,55 mg/mL for 3 strains) and shell (MIC 0,6 mg/mL for 3 strains). These extracts also presented the highest inhibition zone in AWD (30 mm, 38 mm and 32 mm, respectively). A. squamosa and A. indica have high antimicrobial potential against C. pseudotuberculosis.


Introduction
The production of small ruminants in the Brazilian Northeast is a very important socio-economic activity, and more than 92% of goats and 65% of sheep of Brazil are in this region (Magalhães, Martins, Holanda & Lucena, 2018). Generally, this production is based on the extensive system, which results in low sanitary and technological investment. This allows the presence of diseases in the herds, and the one with higher prevalence at Brazilian semiarid is caseous lymphadenitis (CL) (Alves, Santiago & Pinheiro, 2007).
CL is an infectious chronic disease and highly contagious, caused by Corynebacterium pseudotuberculosis, and generates granulomas in external and internal lymph nodes, and can spread through the viscera, which entails economic losses due to the devaluation of meat, skin, milk, and wool (Carminati et al., 2003). Diagnosis often occurs at the later stage of the CL, when the clinical signs are apparent, so, the treatment is generally not effective, which justifies research to discover new therapeutic molecules (Galvão et al., 2017).
The use of medicinal plants is a cosmopolitan practice and the constant studies of phytotherapeutic agents are a good methodology to discover new drugs, especially in tropical regions with abundant biodiversity, for example, Brazil (Araujo et al., 2014). Due to the indiscriminate use of antibiotics, several bacteria have developed mechanisms of resistance to most of the antibiotics, so, it is necessary to find new active principles (Silva, Antunes & Catão, 2011).
Natural antibiotics are extracted from medicinal plants and generally have a broad spectrum, besides having a greater molecular diversity than the synthetic compounds (Delfani, Bahmani, Mohammadrezaei-Khorramabadi & Rafieian-Kopaei, 2017). Several studies have already shown the positive effects on the use of these natural substances, and with the publication of these beneficial factors, the study of plants becomes more present in laboratories routine (Fasihzadeh, Lorigooini & Jivad, 2016;Samani et al., 2016).
Researches have shown several medicinal effects of Annona squamosa, and have clarified that their flavonoids have antibiotic action (Kotkar et al., 2001). Of all the compounds already discovered by Azadirachta indica, 45% are part of the triterpenoids, which has a proven antimicrobial, antitumor, and anti-inflammatory potential (Chen et al., 2018).
Allium sativum has as main compound allicin, which is responsible for its high antibiotic potential, as well as antifungal, antiviral and antiparasitic (Johnson, Olaleye & Kolawole, 2016). The therapeutic properties of Prosopis juliflora tree have already been demonstrated in a variety of in vitro studies, including antimicrobial, anti-inflammatory and antifungal activity (Nagalakshmi & Anuradha, 2017). Research has proven the antioxidant, antibacterial, analgesic and healing effects of Portulaca oleracea, and is listed by the World Health Organization (WHO) as one of the most used herbal plants (Iranshahy et al., 2017;Peng et al., 2015).
The use of plants with antimicrobial potential and present in Brazilian Northeast to combat CL can establish a new treatment with better efficiency and accessibility. For this reason, the aim of this study was to evaluate the in vitro antimicrobial activity of Annona squamosa, Azadirachta indica, Allium sativum, Prosopis juliflora, and Portulaca oleracea, against C. pseudotuberculosis.

Material and Methods
This is a laboratory research developed under controlled conditions with quantitative approach. It was based on experiments with defined methodologies on the antimicrobial effect of plant extracts against C. pseudotuberculosis (Pereira, Shitsuka, Parreira & Shitsuka, 2018).

Plant materials and extraction
The extraction process was based on the methodology of Benfatti, Cordova, Guedes, Magina and Cordova (2010) Table 1. Source: Authors.

Bacterial strains and growth conditions
It was selected three C. pseudotuberculosis isolates, the 76 strain (CAPJ4) -strong biofilm producer -and the 21 strain (CAP3W) -non-biofilm producer -deposited in the GenBank under accession number CP026499 and CP026500, respectively.
Also, it was selected the VD57 strain, deposited in the GenBank under the accession number CP009927. The inoculum was prepared through the transfer of five C. pseudotuberculosis colonies -cultivated in Petri dishes with Brain Heart Infusion agar (BHI, Sigma-Aldrich, St. Louis, USA) -to 5 mL of sterile saline solution 0,9%. After this was measured in a spectrophotometer (600 nm) and adjusted to achieve optical density between 0,08 e 0,1 (~1,5x10 8 UFC/mL).

Antimicrobial sensitivity test
For agar well diffusion assay (AWD) Petri dishes (60x15 mm) were prepared with BHI agar (25 mL) with 4 equidistant holes (6 mm Ø). The inoculum (~1,5x10 8 UFC/mL) was seeded with swab all over the surface and the wells were filled with 20 µL of each extract. The control was ethanol 70%, DMSO 10% and antibiotic (Streptomycin 1 mg/mL + Penicillin -1000 units/mL). The plates were incubated at 37 °C (48 h), and the inhibition zone was measured in millimeters. The analysis of the results followed the CLSI (2018) recommendations with adaptations.
Broth microdilution was performed according to Santos et al. (2019)

Statistical analysis
The results obtained in the AWD assays were submitted to analysis of variance (ANOVA) and it was used as a posttest of multiple-comparison the Tukey's test, with a significance of 5%. Statistical analyses were performed using GraphPad Prism v. 7.05.

Results and Discussion
The results of antimicrobial activity of plant extracts against different strains of Corynebacterium pseudotuberculosis are shown in Tables 2 and 3.
The parts of A. squamosa obtained distinct results in the antibacterial action. Although the ethanolic fraction of A.
squamosa seed has an effect against 21 strain (MIC and MBC 22 mg/mL) and VD57 strain (MIC and MBC 11 mg/mL), the concentrations were high, besides that the DMSO fraction of this extract had no antibacterial effect (Table 2). In opposition to the seed, the other parts of A. squamosa in an ethanolic solvent, demonstrated a satisfactory effect on the tested strains. The stalk had the same MBC for the 3 strains (0,55 mg/mL) and the shell presented the lowest MBC against 21 and VD57 strains (0,6 mg/mL), while the leaf had the best MBC against VD57 strain (3,5 mg/mL). This result corroborates a study that demonstrated that while the methanolic extract of A. squamosa seed had no antimicrobial activity against Staphylococcus aureus, Klebsiella pneumoniae and Enterococcus faecalis, the extract of A. squamosa leaf was effective against these bacteria (Pinto et al., 2017).
The difference between the parts of A. squamosa is also verified in AWD assay (Table 3), in which, against VD57 strain the ethanolic extract of stalk, shell, and leaf of A. squamosa presented inhibition zone of 38 mm, 32 mm e 25 mm, respectively, which had no statistical difference with the antibiotic, while the A. squamosa seed had no inhibition zone.

DMSO 10%
Annona squamosa Although both extracts of A. indica leaf demonstrate anti-Corynebacterium effect, the ethanolic fraction, which reached MIC of 0,12 mg/mL against VD57 and 21 strains, had more satisfactory results when compared to the extract in DMSO 10% (Table 1). A. indica leaf ethanol extract also presented satisfactory results in AWD assay, with inhibition zone of 30 mm (VD57 strain), 20 mm (21 strain) and 23 mm (76 strain), without significant statistical difference compared to the antibiotic, as shown in table 3.
The best antimicrobial action of ethanolic extract compared to DMSO was also observed in A. indica stalk, once in AWD assay, the ethanolic extract obtained inhibition zone (16 mm, 17 mm e 25 mm against 21, 76 and VD57 strains, respectively), while DMSO extract had no antibacterial effect (Table 3). As in this study, Success et al. (2017)  It is observed in Table 2 that P. juliflora presented antimicrobial activity in ethanolic extract (MBC against 21 -0,4 mg/mL) and in DMSO extract (MBC against VD57 -1,3 mg/mL). Despite this, the ethanolic extract had superior antimicrobial activity, which corroborates with a study that indicated that ethanolic and aqueous extracts are better to concentrate active metabolites of P. juliflora (Khandelwal, Sharma & Agrawal, 2016). Care should be taken with this plant because contains neurotoxic piperidine alkaloids (Silva, Silva, Silva & Costa, 2018). Research, Society andDevelopment, v. 10, n. 11, e509101119875, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19875 6 The ethanolic extract of P. oleracea presented antimicrobial activity (Table 2) against all strains (MIC 2,2 mg/mL to 21 and 4,4 mg/mL to VD57 and 76). The study of Peng et al. (2015) demonstrated that the ethanol extract of this plant was the most efficient against S. aureus, Streptococcus agalactiae and Streptococcus dysgalactiae from mastitis in cows. Table 3. Inhibition zone (mm) in agar well diffusion assay (AWD) of selected extracts against three C. pseudotuberculosis strains.

Ethanol 70%
Annona squamosa In all methods of evaluation of antimicrobial activity, the ethanolic fraction showed a better anti-Corynebacterium potential when compared to DMSO (Table 2 and 3). This corroborates with studies that demonstrated that ethanol is superior when compared to solvents such as hexane, chloroform, ethyl acetate, butanol, and petroleum ether (Arya, Thakur & Kashyap, 2012;Hossain & Shah, 2015). Although some studies indicate that DMSO acts on cell membranes, this solvent did not interfere in the results of this study, once the control DMSO 10% did not exert an antibacterial effect, as well as the control ethanol 70% (Cheng, Song, Pas, Meijer & Han, 2015). The antibiotic control presented superior antibacterial efficacy in both tests (Tables 2   and 3) because it has a high concentration of metabolites and the active principle is purified.
Strain 76 had a higher MBC mean (13,2 mg/mL) compared to the VD57 (7,9 mg/mL) and 21 (5,6 mg/mL) strains, probably due to biofilm production that helps in agent survival under adverse conditions (McCarthy et al., 2015). Still, 8 extracts presented antibacterial effect against strain 76, like ethanolic fraction of A. squamosa leaf (MBC 14 mg/mL), that is according to research of Pinto et al. (2017) in which the methanolic extract of A. squamosa leaf induced significant disruption of the biofilm of S. aureus and K. pneumonia.

Conclusion
Seven extracts had antimicrobial activity against three strains -six ethanolic fractions (A. squamosa stalk, shell, and leaf; A. indica leaf; P. juliflora; P. oleracea) and one fraction in DMSO (P. juliflora). Thus, these phytotherapics have potential action against different strains of C. pseudotuberculosis, relevant for CL treatment, due to the variability of strains found in the herds. The extracts of A. squamosa, A. indica, and P. juliflora demonstrated higher antimicrobial potential against C.

pseudotuberculosis.
Future researches to identify the components of these plants that demonstrated in vitro antimicrobial potential against C. pseudotuberculosis are important to understand the mechanisms of action of extracts and isolated compounds. In addition, studies in the animal model must be developed to evaluate the antibacterial activity in vivo and verify possible toxic effects.