Inhibitory effect of Persea cordata Mez. (pau-andrade) bark extracts against Clostridium perfringens causing gangrenous mastitis
Efecto inhibitorio de extractos de corteza de Persea cordata Mez. (pau-andrade) contra Clostridium perfringens causa de mastitis gangrenosa
Valfredo Schlemper, Susana Regina de Mello Schlemper, Denise Maria Sousa de Mello
Universidade Federal da Fronteira Sul (UFFS), Campus Realeza, PR, Brazil.
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ABSTRACT
Introduction: Gangrenous mastitis is a special clinical presentation of mastitis in cattle and small ruminants. The bark of the tree Persea cordata Mez. is used in Brazilian ethnoveterinary medicine to treat wounds in farm animals.
Objectives: Examine in vitro antimicrobial action of apolar fractions of P. cordata bark against a wild strain of C. perfringens isolated from the udder of a cow with gangrenous mastitis, and against a reference strain.
Methods: A milk sample was collected from the udder, aliquots were diluted and Gram-stained smears were performed. The aliquots were inoculated in broth and planted in blood agar, and then incubated in anaerobiosis at 37oC / 24h. Biochemical identification was based on bacterial isolation. In vitro inhibitory activity of apolar fractions of P. cordata was evaluated by agar diffusion and MIC (minimum inhibitory concentration) using the agar dilution method.
Results: In both tests the plant extracts displayed significant in vitro inhibitory activity against the clinical and reference strains of C. perfringens assayed.
Conclusion: The study is the first demonstration of the inhibitory effect of P. cordata on C. perfringens, due to its antimicrobial properties, which serves as evidence supporting its folk use. The extracts could be used as coadjuvants in the treatment of gangrenous mastitis.
Keywords: medicinal plants; Persea cordata; antimicrobial; Clostridium perfringens; mastitis.
Introducción: la mastitis gangrenosa es una presentación clínica especial de mastitis en el ganado y pequeños rumiantes. Persea cordata Mez. , es un árbol conocido en la etnoveterinaria brasileña, cuya corteza se utiliza en la curación de heridas en animales de granja.
Objetivos: investigar el efecto antimicrobiano in vitro de fracciones apolares de la corteza de P. cordata contra una cepa salvaje de C. perfringens, aislada de la ubre de una vaca con mastitis gangrenosa y una cepa de referencia.
Métodos: Se recogió una muestra de leche de la ubre, se diluyeron alícuotas, y se realizaron frotis teñidos por Gram. Las alícuotas fueron inoculadas en caldo y sembradas en agar sangre, y posteriormente incubados en anaerobiosis a 37 oC/24h. La identificación bioquímica fue realizada a partir del aislamiento bacteriano. La actividad inhibitoria in vitro de las fracciones apolares de P. cordata fue evaluada utilizando la técnica de difusión en agar y la CMI (concentración minima inhibitoria) mediante el método de dilución en agar.
Resultados: los extractos de la planta, en ambas pruebas, presentaron significativa actividad inhibitoria in vitro contra las cepas clínica y de referencia de C. perfringens ensayadas.
Conclusión: se concluyó que, por primera vez, se demuestra un efecto inhibitorio de P. cordata sobre C. perfringens, reforzando el uso popular, debido a sus propiedades antimicrobianas. Los extractos podrán ser utilizados como coadyuvantes en el tratamiento de la mastitis gangrenosa.
Palabras clave: plantas medicinales; Persea cordata; antimicrobiano; Clostridium perfringens; mastitis.
INTRODUCTION
]]> Mastitis is the inflammation of breast tissue caused by bacteria, viruses, algae and fungi,1 enzootic in bovine what causes important loss to the milk-production chain. Gangrenous mastitis is a special clinic feature of mastitis in bovine, caused mainly by Staphylococcus aureus, Mannheimia haemolytica, Escherichia coli, Clostridium perfringens and Arcanobacterium pyogenes.2 Published reports about the occurrence of gangrenous mastitis by C. perfringens are relatively old.3-5 In Brazil, the descriptions of gangrenous mastitis by C. perfringens in bovine are rare. Most of the studies have described the illness in small ruminants.6-8 Baldassi9 related the occurrence of gangrenous mastitis in bovine caused by Clostridium species and Gonçalves10 published the first gangrenous mastitis clinical case in Brazil caused C. perfringens in bovine. Although most of bacterial mastitis are caused by aerobic ones,11 there is growing concern of the involvement of anaerobic organisms in those pathological processes. The main reason for the limited number of published studies on the involvement of Clostridium sp in mastitis is due to the difficulty of microorganisms growing.12 Gangrenous mastitis by C. perfringens is caused, predominantly, by the upward contamination of the teat canal, because a bacterium is frequently isolated of faeces from healthy animals13 and the environmental contamination supported by weather conditions and organic matter accumulation in areas where animals live can propitiate upward contamination of breast papillae. Moreover, before the estimated date of birth, cows show weakened immune system due to colostrogenesis and the release of endogenous corticoids,14 features which can support contamination and installment of the infectious process. Local alterations, as lack of oxygen in injuries and digestive tract, so propitiating tissues invasions and their proliferation.15 Clinically, it is characterized by mammary gland redness and necrosis of parenchyma accompanied with bloody, foul-smelling milk.In searching for therapeutic alternatives against bacteria, medicinal plants have been standing out as potential sources of active ingredients able to control infectious processes. To that end, given the occurrence of gangrenous mastitis in bovine in Southwest Paraná, this study was based on the search for plants with ethnopharmacological indicatives for infections in animals.
Persea cordata (Vell.) Mez. (Lauraceae) a common big tree in Araucaria Forest, also known as "pau-andrade", "abacateiro do mato", "maçaranduba" or "canela rosada"16 in popular medicine, has its bark used as plaster for wound healing.17-18 It is common, in farms in the South Brazil, to find P. cordata with their barks removed carefully in order to treat some diseases in animals. Some biological activities attributed toP. cordata barks have already been proved experimentallyin vivo and in vitro such, antibacterial and antifungal,19,17 antispasmodic.20,21 neuroprotector22 and as antiedematogenic18 actions. P. cordata healing action was described from the use of an ointment made of its barks,23 so indicating a beneficial effect on granulation tissue evolution, facilitating the newly epithelial tissue and reducing time of wounds mobilization. Possibly, contributing with its healing action, it was found its polar fraction of ethyl acetate showed antimicrobial activity against Staphylococcus aureus and Streptococcus pyogenes, which corroborate with its popular use in rural areas for treating infected wounds of animals.19,23
Antibacterial activity of the plant was related against Gram-positive S. aureus, S. epidermidis and Bacillus cereus bacteria, and against Gram-negativePseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli,Shigella dysenteriae, Proteus vulgaris and Salmonella typhimurium.19,23 The objective of this study was to evaluate the inhibitory activity of P. cordata semi-purified extracts in vitro on the growing of C. perfringens, an anaerobic bacteria causing gangrenous mastitis.
METHODS
Botanical material
P. cordata barks were collected in Fazenda Leny, Bom Retiro, Santa Catarina, according to the agreement of the farmer and where it was inserted the legal forest reserve. Intervention in the natural environment was minimal and did not cause risk for the integrity of the trees. For the barks collection, the project was registered in Plataforma Chico Mendes (SISBIO) under the number 34821-1. The botanical material was identified by Dr. Roseli Botoluzzi (Agronomy Graduation Course/ CAV/ UDESC, Lages - SC) and a dried specimen was set in the herbarium of the Centro Agroveterinário, Lages, under the number 045.
Extract and fractions collection
Phytochemical screening
Chromatographic profile of P. cordata methanolic extract was evaluated through thin-layer chromatography (TLC) using aluminum chromatographic plates, pre-coated with thin layers of silica (Merck, 200 µm thickness) with different solvent systems. The research of bioactive secondary metabolites was performed through Liebermann-Buchard reaction for terpenes and steroids, Keller-Kelianni and Baljet reactions for aglycone and total sugars detection, Mayer and Bertrand, Bouchardat and Dragendorff reactions for alkaloids.24,25
Microorganisms, isolation and identification
Bacterial samples used in the study were: a strain of the material collected from the udder of a Holstein cow, 4 years-old, with clinical history of gangrenous mastitis; and a standard C. perfringens strain (ATCC 13124) used as reference.
At clinical examination, the veterinarian who attended the animal related that it showed sudden lactation drop, anorexia, hyperthermia, continuous decubitus and melena. The front right quarter of the udder was swollen, touch-sensitive and blackened, and the milk secretion showed serous fluid with yellowish flocculation and bloody striations. Milk samples from the affected udder quarter were aseptically collected in sterile tubes with screw caps. At laboratory, aliquots were diluted in 0.5 mL of 0.9 % saline solution and from those it was performed stained smears by Gram technique.
Both samples (collected material and standard one) were inoculated in tubes with Cooked Meat Medium (CMM, Difco). Tubes were previously heated at 80 ºC during 15 min in water bath and quickly cooled in tap water, and they were incubated at 37 ºC for 24-48 h. An aliquot of each sample was inoculated by pouring into petri dishes with 5 % sheep blood agar, and incubated under aerobiosis and strict anaerobiosis in McIntosh & Fields jars at 37 ºC during 24-48 h. After incubation, the colonies were observed in appearance, coloring and haemolysis. Samples showing doubled-halo, smooth, rounded and umbilical hemolytic colonies were submitted to Gram stain, and in sequence they were inoculated in CMM and incubated at 37 ºC for 24-48 h.
With the resulting growth of the broth it was performed standard biochemical assays in order to identify the species: sugars fermentation (glucose, lactose, sucrose, maltose, salicin and mannitol), lipase and urease activity, indole production, hydrolysis gelatin, casein digestion, nitrate reduction and iron-milk reaction,8,26,27 Additionally, it was made subcultures into blood agar plates and agar base tryptose sulfite cycloserin (TSC) plus 5 % egg-yolk selective-indicator media to demonstrate double zone of beta hemolysis and lecithinase reaction, respectively, and incubated at 37 ºC for 18-24 h.
]]>Preliminary screening
The inhibitory activity of semi-purified hexane and dichloromethane fractions from P. cordata barks was tested against standard and collected strains through agar diffusion method.8 Strains were activated into thioglycollate broth and a 106 CFU/mL final inoculum had its density adjusted by 530 nm spectrophotometer. In the sequence, they were inoculated into 5 % sheep blood agar plates where previously it was made wells at 6 mm diameter into a circular layout parallel to the edge of the plates with a cylindrical metal device, sterilized and with sharp edges. Wells were filled with 50 µL P. cordata extracts in 500, 1000 and 2000 µg/mL concentrations. Then, plates were incubated at 37 ºC for 48 h in bacteriological incubator under anaerobiosis. After that, plates were examined for the increase of colonies. Results were collected according to Bauer et al,28 and growth inhibition was analyzed through measurement of halos with pachymeter. Antibacterial activity was expressed through concentration-response curves of bacterial growth inhibition. As a positive control it was used commercial antibiotics in liquid form, as penicillin (10 µg/mL). Plates were incubated for 24-48 h at 37 ºC into anaerobic chamber.
Minimum inhibitory concentration
Minimum inhibitory concentration (MIC) of P. cordata crude extract and semi-purified fractions on C. perfringens growing was determined through broth macrodilution method protocol29 in tubes with Reinforced Clostridial Medium (RCM) (Labor M443). MIC was defined as the smallest concentration of each extract which visibly inhibited bacterial growing. To this end, a battery of tests using 9 tubes with different concentrations of each extract fraction and crude extract (1 a 256 µg/mL) was prepared in final volume from 1 to 2 mL per tube. An inoculum-control tube was prepared as well. Tubes with extract fractions were inoculated with a 5 × 106 UFC/mL standard bacterial medium. Final cell concentration was adjusted using spectrophotometer. After 24-48 hours of incubation in anaerobiosis chamber, it was performed the turbidimetry process according to Daguet and Chabbert30 technique. The determination of penicillin MIC for the reference strain was done as collateral control for the validation of the methodology.
Statistical analysis
The results were contained with the average of the experiments ± standard error of the averages, except IC50s (i.e. the concentration of the extracts requested to inhibit the bacteria growth 50 % in relation to answer controls), which are presented as geometric means accompanied by their respective 95 % confidence intervals. The statistical analyses were obtained by the ANOVA test, followed by the Dunnett's test when necessary. p< 0.05 or p< 0.01 was considered significant. The IC50s were obtained using the GraphPad Prism 7.0 program.
]]>RESULTS
Milk aliquots smear from collected samples showed short gram-positive bacilli. On blood-agar under anaerobiosis, colonies were gray-white color, big, smooth, rounded, regular convex, surrounded by a typical zone of β-haemolysis, and stained smear showed gram-positive bacillus. In biochemical tests were founded: fermentation of sugars (positive for glucose, lactose, sucrose, maltose and salicin; negative for mannitol, a negative activity on lipase and urease, no indol production, gelatin hydrolysis, casein digestion, reduction of nitrates in nitrites and promotion of stormy clot.8 Additional cultivation in blood agar and TSC agar showed double haemolysis zone and lecithinase positive reaction, respectively. Based on phenotypic and biochemical characteristics, the isolated microorganism was identified as C. perfringens.
When examined in agar-diffusion test, it was observed both hexane (fig. 1A and 1B) and dichloromethane (fig. 1C and 1D) fractions of P. cordata caused significant and dependent-concentration inhibition halos as for control strains as for the collected ones in relation to control groups. It was calculated fractions potencies based on two triplicate samples for each experimental protocol. IC50s obtained from hexane fraction were 1560 (1270/1866) µg/mL for each reference strain (fig. 1A) and 467(444/509) µg/mL for collected strain (fig. 1B). For dichloromethane fraction, IC50s potencies ranged 648 (591/689) µg/mL for reference strain (fig. 1C) and 482 (433/499) µg/mL for collected strain (fig. 1D). For experiments using hexane and dichloromethane fractions for reference strain, the greatest tested concentration (2000 µg/mL) had inhibition efficacy up to the concentration of positive control (G penicillin, 10 µg/mL), and for the collected strain there was a greater efficacy for dichloromethane fraction in relation to positive control.
In broth macrodilution test, the semi-purified fraction of hexane showed moderate bacteriostatic activity with MIC values in an interval of 0.5 to 2 µg/mL against reference and collected strains (table). For dichloromethane fraction, MIC values ranged around 16 µg/mL for both strains. But the crude extract was less potent than semi-purified fractions showing MIC values of 32 µg/mL for clinical strain and 64 µg/mL for reference one.
DISCUSSION
Infectious diseases caused by Clostridium sp. have a broad range of clinical severity that varies greatly. Among clostridium, C. perfringens has been associated with sudden death, toxicity, bleeding and gastrointestinal diseases.31,32,33 However, mastitis caused by C. perfringens is a disease that generally shows a dramatic clinical condition with irreversible damage to mammary gland of female bovine and ovine in high dairy production.24,34 There is a huge quantity of publications about antibacterial activity of medicinal plants around the world,35-37 and a lot of plants have been researched due their biological activity against C. perfringens,38-41 including clinical strains from isolated infections of hospitalized humans42,43 or C. perfringens isolated from commercial broiler chickens diagnosed with necrotic enteritis,44,45 but not with clinical pathogenic strains isolated from udder infection of lactating dairy cows. Researches performed with crude extracts of several plants and fungi showed, in a sorting of 238 extracts, 25 ones showing antimicrobial activity against C. perfringens.40 Bento et al.45 analyzed the synergism of essential oils of oregano, rosemary and thyme and its bactericidal effect to confront C. perfringens from standard strains. Jimoh et al.46 obtained important results with garlic against C. perfringens from caeca of broiler chickens. Until now, there is no reference in the literature regarding the use of P. cordata extracts or fractions against C. perfringens.
]]> P. cordata is a Brazilian tree from Araucaria Forest bioma which has ethno-indication as medicinal plant for treating dermatological infection and inflammation, probably due its action against anaerobic and aerobic skin bacteria as C. perfringens. While we were performing experiments with P. cordata against bacterial strains of veterinary interest, we had the opportunity to receive a clinical material of mammary infection which was confirmed clinically and microbiologically as gangrenous mastitis by C. perfringens. In this study, for the first time, it was demonstrated a significant inhibitory effect of P. cordata fractions against wild anaerobic microorganisms that usually have high resistance to conventional antibiotics.47There was proposed a classification for the antimicrobial activity of vegetal materials, based on CIM results, as follow: strong inhibitor - CIM until 0.5 mg/mL; moderate inhibitor - CIM between 0.6 and 1.5 mg/mL; light inhibitor - CIM up to 1.6 mg/mL.48 In other studies the inhibition of vegetal concentrations until 2 mg/mL characterized the extracts as potentially antimicrobial.49
In relation to the tests utilized, the hexane extract of P. cordata in broth macrodilution test, for all dilutions showed inhibitory effect in concentrations smaller than 1 µg/mL for standard strain and smaller than 2 µg/mL for clinical strain of C. perfringens. Dichloromethane extract inhibited bacterial growing significantly, reducing turbidity in the concentration of 16 µg/mL for both samples. Turbidity inhibition with the crude extract was around 32 µg/mL for collected sample and 64 µg/mL for standard sample, so demonstrating less power than semi-purified fractions.
Phytochemical studies by Sieben50 showed among secondary metabolites obtained through hexane and dichloromethane fractions founded in P. major barks (P. cordata) are pointed out terpenoids, steroids, aglycons and total sugars, coinciding with we found in our fractions with thin-layer chromatography. Among them, triterpenes and steroids are predominant in the plant and they are highlighted as traditional phytochemical compounds with a well-established antimicrobial activity in other medicinal plants and which are efficient against different gram-positive and gram-negative microorganisms51,52 including Clostridium spp.53,54 As those steroids and triterpenes are predominant in less polar fractions as hexane and ethyl acetate fraction to a lesser extent, antibacterial actions of the plant could be attributed to those active principles.
Within the classification proposed, hexane extract of P. cordata barks showed antimicrobial action with a strong inhibitor against C . perfringens, in contrast to the reference antibiotic used (penicillin). The findings have suggested that less polar P. cordata fractions show efficient phytochemical compounds for inhibiting the growth of C. perfringens, this way justifying the popular use of the plant to treat infirmities caused by that microorganism.
We have demonstrated an important antiedematogenic effect of P. cordata polar fractions,18 that in connection with its antibacterial effect17 could contribute to the healing actions of the plant23 according to ethnopharmacological findings from popular medicine. Thus, as a phytotherapic product, P. cordata barks can be a multifunctional medicine to treat skin wounds of animals, including infected ones. The studied P. cordata showed inhibitory activity for the anaerobic bacteria C. perfringens which causes several infectious pathologies associated to necrosis and hemorrhagic signs on the udder skin.55 Despite being a fecal indicator of food contamination, it can be isolated after previous contamination to a distant source due to its ability to transform itself from a vegetative state into a microorganism which produces resistant spores, depending on environmental conditions.56 Plants with antibacterial activity can potentially be used to treat diseases caused by bacterial infections. In popular medicine, the treatment of infected wounds is an indicative of that activity. Due to the growing of C. perfringens strains more resistant to conventional drugs, new antibiotics will be needed for the therapy of animal infections in the future. There is an ecological rationality that antibacterial cytotoxic products should be synthesized in plants again after microbial attack in order to protect plants from pathogens.47 Thus, antimicrobial compounds naturally derived from plants can be a new prospect to fight against multi-drug resistant pathogens.
We have concluded that, for the first time, it was demonstrated an inhibitory effect of P. cordata extracts on C. perfringens, reinforcing the popular use of P. cordata due to its antimicrobial properties. Thus the extracts may be used as coadjuvants in the treatment of gangrenous mastitis.
ACKNOWLEDGEMENTS
The authors thank PIBIC-CNPq for financial support. Authors are grateful to Dr. Alan Lara Nenmaier for technical support and provision of milk sample.
]]>The author has no conflicts of interest.
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Recibido: 11 de septiembre de 2015.
Aprobado: 4 de noviembre de 2016.
Valfredo Schlemper. Universidade Federal da Fronteira Sul, Brazil. Correo electrónico: valfredo.schlemper@uffs.edu.br
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