Vibrio spp. from Macrobrachium amazonicum prawn farming are inhibited by Moringa oleifera extracts

Raimunda Samia Nogueira Brilhante， Jamille Alencar Sales， Celia Maria de Souza Sampaio，F(xiàn)rancisco Geraldo Barbosa， Manoel de Araújo Neto Paiva， Glaucia Morgana de Melo Guedes，Lucas Pereira de Alencar， Yago Brito de Ponte， Tereza de Jesus Pinheiro Gomes Bandeira，José Luciano Bezerra Moreira， Débora de Souza Collares Maia Castelo-Branco， Waldemiro de Aquino Pereira-Neto， Rossana de Aguiar Cordeiro， José Júlio Costa Sidrim， Marcos Fábio Gadelha Rocha，

1Department of Pathology and Legal Medicine， Postgraduate Program in Medical Microbiology， Specialized Medical Mycology Center， Federal University of Ceará， Fortaleza， Ceará， Brazil

2School of Veterinary Medicine， Postgraduate Program in Veterinary Sciences， State University of Ceará， Fortaleza， Ceará， Brazil

3Department of Organic and Inorganic Chemistry， Federal University of Ceará， Fortaleza， Ceará， Brazil

Vibrio spp. from Macrobrachium amazonicum prawn farming are inhibited by Moringa oleifera extracts

Raimunda Samia Nogueira Brilhante1*， Jamille Alencar Sales2， Celia Maria de Souza Sampaio2，F(xiàn)rancisco Geraldo Barbosa3， Manoel de Araújo Neto Paiva2， Glaucia Morgana de Melo Guedes1，Lucas Pereira de Alencar2， Yago Brito de Ponte2， Tereza de Jesus Pinheiro Gomes Bandeira1，José Luciano Bezerra Moreira1， Débora de Souza Collares Maia Castelo-Branco1， Waldemiro de Aquino Pereira-Neto1， Rossana de Aguiar Cordeiro1， José Júlio Costa Sidrim1， Marcos Fábio Gadelha Rocha1，2

1Department of Pathology and Legal Medicine， Postgraduate Program in Medical Microbiology， Specialized Medical Mycology Center， Federal University of Ceará， Fortaleza， Ceará， Brazil

2School of Veterinary Medicine， Postgraduate Program in Veterinary Sciences， State University of Ceará， Fortaleza， Ceará， Brazil

3Department of Organic and Inorganic Chemistry， Federal University of Ceará， Fortaleza， Ceará， Brazil

ARTICLE INFO

Prawn

Macrobrachium amazonicum

Moringa oleifera

Vibrio

Antimicrobial

Objective： To investigate the in vitro antimicrobial potential of extracts of stem， leaves，flowers， pods and seeds of Moringa oleifera （M. oleifera） against Vibrio spp. from hatchery water and the prawn Macrobrachium amazonicum. Methods： The ethanol extracts of stem， leaves，pods and seeds and chloroform extract of flowers of M. oleifera were tested against Vibrio cholerae （V. cholerae） serogroups non-O1/non-O139 （n=4）， Vibrio vulnificus （n=1） and Vibrio mimicus （n=1）. Escherichia coli （E. coli） （ATCC? 25922） was used as quality control. Vibrio species were obtained from Macrobrachium amazonicum prawns and from hatchery water from prawn farming. The Minimum Inhibitory Concentration （MIC） was determined by broth microdilution method. Results： The best result was obtained with the ethanol extract of pods，which inhibited three strains of the V. cholerae， Vibrio vulnificus， Vibrio mimicus and E. coli（MIC range 0.312-5.000 mg/mL）. The chloroform extract of flowers was effective against all V. cholerae strains and E. coli （MIC range 0.625-1.250 mg/mL）. However， the ethanol extracts of stem and seeds showed low effectiveness in inhibiting the bacterial growth. Conclusions：The extracts of pods， flowers and leaves of M. oleifera have potential for the control of Vibrio spp. Further studies are necessary to isolate the bioactive compounds responsible for this antimicrobial activity.

Document heading doi：10.1016/j.apjtm.2015.10.012

1. Introduction

The cultivation of shrimp can be threatened by diseases causedby Vibrio species， which can result in up to 100% mortality， 24 h after the appearance of infection［1］. Vibrio cholerae （V. cholerae），Vibrio mimicus （V. mimicus） and Vibrio vulnificus （V. vulnificus）are opportunistic pathogens capable of causing lethal infections in farmed crustaceans when there are stressful environmental conditions， nutritional imbalance and predisposing lesions［2］. Moreover， antimicrobial resistance in these microorganisms has been observed［3］.

The emergence of antibiotic resistant bacteria has driven research to find new compounds with antimicrobial properties in plants usedin traditional medicine， such as Moringa oleifera （M. oleifera） （Lam.）［4-10］.

M. oleifera is a well-known and widely distributed tree species，belonging to the family Moringaceae［11］. In Brazil it can be found in the Northeast， mainly in the states of Maranh?o， Piauí and Ceará［9］. The antimicrobial properties of M. oleifera have been attributed to different parts of the plant， such as leaves， flowers， seeds， pods and stems［9,12,13］. The literature reports the antimicrobial potential of Moringa against bacteria and fungi isolated from shrimp farming［5,9］. Thus， the objective of this study was to evaluate the in vitro antimicrobial potential of extracts of stem， leaves， flowers， pods and seeds of M. oleifera against Vibrio species isolated from hatchery water and Macrobrachium amazonicum （M. amazonicum） prawn.

2. Materials and methods

2.1. Extracts

The extracts were obtained from specimens of M. oleifera grown in Fortaleza， Ceará， Brazil， and provided by the Laboratory of Applied Phytochemistry， Federal University of Ceará. Stem， leaves， pods and seeds were dried in a heated chamber at 40 ℃ and then subjected to three successive extractions by cold maceration with ethanol at intervals of 24 h， originating the ethanol extracts， while flowers were dried at 40 ℃ and then subjected to three successive extractions by cold maceration with chloroform at intervals of 24 h， originating the chloroform extract. After filtration， the respective solvents were evaporated under reduced pressure in a rotary evaporator， leaving only the concentrated constituents extracted from the plant parts［7］.

2.2. Strains of Vibrio spp.

V. cholerae serogroups non-O1/non-O139 （n=4）， V. mimicus（n=1） and V. vulnificus （n=1）， belonging to the bacterial collection of the Laboratory of Emerging and Reemerging Pathogens of Ceará Federal University， were used in this study. These strains were obtained through the collection of specimens of ovigerous M. amazonicum females from Sapiranga Lake （3048'3.46” S and 38027'30.83” W）， Fortaleza， Ceará， Brazil， and samples of hatchery water from M. amazonicum farming， during the larval development stage， at the Laboratory of Shrimp Farming of the State University of Ceará.

2.3. In vitro susceptibility test

The in vitro susceptibility test with extracts of M. oleifera was performed following the method described by Rocha［7］ in 2011， with some modifications. Initially， each extract was dissolved in dimethyl sulfoxide （DMSO） （LGC Biotecnologia Ltda， S?o Paulo， Brazil）and then diluted in Müeller-Hinton broth （Difco?， S?o Paulo，Brazil）. In previous tests with strains of Vibrio spp.， we verified that DMSO alone at concentrations up to 5% was not able to inhibit the growth of these strains. Thus， the concentration of DMSO in the susceptibility test did not exceed 5%， to assure that results refer to the action of each extract tested［7］.

Minimum inhibitory concentration （MIC） of M. oleifera extracts against strains of Vibrio spp. was determined through the broth microdilution method as standardized by the Clinical Laboratory Standards Institute， based on the document M07-A9［14］. The MIC was considered the lowest concentration of the extracts able to inhibit 50% of bacterial growth compared to the control growth［9］. The strain Escherichia coli （E. coli） （ATCC? 25922） was included as quality control. The initial concentration of each extract used was 20 mg/mL and the range of concentrations evaluated in the susceptibility test was from 0.01 to 5.00 mg/mL. The microdilution assays were performed in 96 well plates with a final volume of 200 μL， incubated at 35 ℃ and read after 20 h， according to the document M45-A2［15］. The inocula were prepared at a turbidity of 0.5 on McFarland scale （108CFU/mL）， and then diluted with Müller-Hinton broth so that each well after inoculation presented approximately 5×105CFU/mL. All assays were performed in duplicate， and for each strain growth control and sterility control of the culture medium were included［14］. The reading was performed with a spectrophotometer （BioteK?， Winooski， United State） at 590 nm and the obtained absorbance values were corrected by the absorbance obtained for each tested extract alone. Only extracts that inhibited the control strain （E. coli ATCC? 25922） were considered as to have antimicrobial activity against Vibrio spp. Chloramphenicol（Sigma-Aldrich? Brazil Ltda， S?o Paulo， Brazil） was used as standard antibiotic， as recommended by the document M100-S22［16］.

2.4. Research licensing

This study was previously approved by the Chico Mendes Institute for Conservation of Biodiversity/Biodiversity Authorization and Information System — SISBIO， under the number 28175-1.

3. Results

The ethanol pod extract showed the best inhibitory activity against isolates from the hatchery water， with MIC values ranging from 0.3125 to 1.250 mg/mL against three strains of V. cholerae non-O1/ non-O139 （3/4） and an MIC of 5 mg/mL against V. vulnificus. This extract also showed inhibitory effect against V. mimicus from the digestive tract of M. amazonicum and E. coli （ATCC? 25922）， with MIC values of 1.25 mg/mL and 2.50 mg/mL， respectively （Table 1）.

Table 1 MIC values of M. oleifera extracts against Vibrio spp. recovered from hatchery water and M. amazonicum prawn.

The chloroform extract of flowers was effective against all strains of V. cholerae non-O1/non-O139， with MIC values ranging from 0.625 to 1.250 mg/mL， as well as against E. coli strain （ATCC? 25922） （MIC 1.25 mg/mL） （Table 1）.

The ethanol extract of leaves， in turn， had inhibitory activity against two strains of V. cholerae non-O1/non-O139 （2/4）， with MIC values of 0.078 and 0.625 mg/mL， and also presented MIC value of 5 mg/ mL against V. mimicus and E. coli （Table 1）.

The ethanol extract of stem and seeds showed low effectiveness in inhibiting the growth of Vibrio spp. strains， as well as no effect against E. coli， as shown in Table 1. The assay performed with the standard antibiotic， chloramphenicol， against the control strain E. coli（ATCC? 25922） presented MIC value within the range established in the document M100-S22 （17） （Table1）.

4. Discussion

Almost all parts of the M. oleifera have multiple industrial and medical uses［17］. The pharmacological potential of this plant has been described in the literature， in particular the antimicrobial activity of the extracts of pods， flowers， leaves， stem and seeds［9,10, 18］. Thus， this study aimed to verify the antimicrobial activity of M. oleifera against Vibrio species， considering that these microorganisms are opportunistic zoonotic pathogens and can cause economical losses to shrimp farming and public health problems， as well［19］.

The results of this study demonstrate the antibacterial activity of extracts from different parts of M. oleifera against Vibrio strains isolated from prawn hatchery water and from M. amazonicum. The antibacterial activity of the extracts of leaves， flowers and pods has been reported in other studies against Gram-negative bacteria［10,11］. In addition， the antimicrobial activity of M. oleifera extracts has also been observed against Candida species isolated from M. amazonicum farming in previous study［9］.

The extracts of pods， flowers and leaves of M. oleifera showed to be more effective， when compared to the other extracts. The ethanol extract of pods presented the best inhibitory activity against species of Vibrio spp.， and it was the only one capable of inhibiting V. vulnificus. The growth of the E. coli strain （ATCC? 25922） was also inhibited by this extract. Arora and Onsare［10］ verified the effectiveness of the pod extract of M. oleifera against the Gramnegative bacteria Klebsiella pneumoniae， Pseudomonas aeruginosa，E. coli and Salmonella typhimurium. However， there are few reports regarding the inhibitory effect of extracts of M. oleifera against Vibrio species.

Some studies on the history of traditional medicine demonstrated the effectiveness of preparations made of leaves， pods， flowers and seeds against microorganisms that cause human infections［17］. Therefore， studies with V. mimicus， V. vulnificus and V. cholerae noncholera serogroup are important， because these bacteria inhabit estuarine and marine environments and have often been associated with sporadic cases of diarrhea， sepsis and infections， after ingestion of seafood or exposure to contaminated aquatic environment［19-21］. In a previous study by Rocha［9］ in 2014， the chloroform extract of flowers presented antifungal activity against Candida spp. and Hortaea werneckii isolated from M. amazonicum farming. Based on the observation of this antimicrobial action， we tested the chloroform extract of flowers against Vibrio species from the same environment. The chloroform extract of flowers also inhibit Vibrio strains from M. amazonicum cultivation water and the control strain. According to Anwar［11］， the antibacterial property of M. oleifera flowers has been attributed to a substance called pterygospermin［22］. In addition to this compound， other substances， such as benzyl glucosinolates and their cognate isothiocyanate extracted from M. oleifera also have antimicrobial properties against bacteria［17］. Therefore， it is worth noting the need to investigate the bioactive compounds of plants that exhibit antimicrobial activity.

The low antimicrobial effectiveness of extracts of seeds and stem of M. oleifera found in this study has also been demonstrated by other authors［9,13,23］. Probably， this low antimicrobial effectiveness can be associated with inherent factors of Vibrio spp. which decrease or neutralize the activity of the bioactive compounds contained in the seeds and stem of M. oleifera［24］.

Thus， the ethanol extracts of pods and leaves and the chloroform extract of flowers of M. oleifera presented potential to control Vibrio spp.， although further research is necessary to determine the bioactive compounds responsible for this antimicrobial activity.

Conflict of interest statement

We declare that we have no conflict ofinterest.

Acknowledgments

This work was supported by grants from the National Council for Scientific and Technological Development （CNPq； Brazil； Processes 445670/2014-2） and Coordination Office for the Improvement of Higher Education Personnel （AEI-0052-000650100/11）.

［1］ Costa RA， Vieira GHF， Silva GC， Vieira RHSF， Sampaio SS. Susceptibilidade 'in vitro' a antimicrobianos de estirpes de Vibrio spp isoladas de camar?es （Litopenaeus vannamei） e de água de cria??o destes animais provenientes de uma fazenda de camar?es no Ceará. Braz J Vet Res An Sci 2008； 45（6）： 458-462.

［2］ Banerjee S， OoI MC， Shariff M， Khatoon H. Antibiotic resistant Salmonella and Vibrio associated with farmed Litopenaeus vannamei. Sci World J 2012； 2012（1）： 130-136.

［3］ Rebou?as RH， Sousa OV， Lima AS， Vasconcelos FR， Carvalho PB， Vieira RHSF. Antimicrobial resistance profile of Vibrio species isolated from marine shrimp farming environments （Litopenaeus vannamei） at Ceará，Brazil. Environ Res 2011； 111（1）： 21-24.

［4］ Chuang PH， Lee CW， Chou JY， Murugan M， Shieh BJ， Chen HM. Antifungal activity of crude extracts and essential oil of Moringa oleifera Lam. Bioresour Technol 2007； 98（1）： 232-236.

［5］ Vieira GHF， Mour?o JA， ?ngelo AM， Costa RA， Vieira RHSF. Antibacterial effect （in vitro） of Moringa oleifera and Annona muricata against Gram positive and Gram negative bacteria. J Inst Trop Med S?o Paulo 2010； 52（3）： 129-132.

［6］ Arun T， Rao PCH. Phytochemical screening and antibacterial activity of Moringa oleifera Lam. against Proteus mirabilis from urinary tract infected patients. Int J PharmTech Res 2011； 3（4）： 2118-2123.

［7］ Rocha MFG， Aguiar FLN， Brilhante RSN， Cordeiro RS， Teixeira CEC，Castelo-Branco DSCM， et al. Extratos de Moringa oleifera e Vernonia sp. sobre Candida albicans e Microsporum canis isolados de c?es e gatos e análise da toxicidade em Artemia sp. Cienc Rural 2011； 41（10）： 1807-1812.

［8］ Prasad TNVKV， Elumalai EK. Biofabrication of Ag nanoparticles using Moringa oleifera leaf extract and their antimicrobial activity. Asian Pac J Trop Biomed 2011； 1（6）： 439-442.

［9］ Rocha MFG， Alencar LP， Brilhante RSN， Sales JA， Ponte YB， Rodrigues PHA， et al. Moringa oleifera inhibits growth of Candida spp. and Hortaea werneckii isolated from Macrobrachium amazonicum prawn farming with a wide margin of safety. Cienc Rural 2014； 44（12）： 1-7.

［10］ Arora DS， Onsare JG. In vitro antimicrobial evaluation and phytoconstituents of Moringa oleifera pod husks. Ind Crops Prod 2014；52（1）： 125-135.

［11］ Anwar F， Latif S， Ashraf M， Gilani AH. Moringa oleifera： A food plant with multiple medicinal uses. Phytother Res 2007； 21（1）： 17-25.

［12］ Ferreira RS， Napole?o TH， Santos AFS， Sá RA， Carneira-da-Cunha MG，Morais MMC， et al. Coagulant and antibacterial activities of the watersoluble seed lectin from Moringa oleifera. Appl Microbiol 2011； 53（2）：186-192.

［13］ Arora DS， Onsare JM， Kuar H. Bioprospecting of Moringa（Moringaceae）： microbiological perspective. J Pharmacogn Phytochem 2013； 1（6）： 193-215.

［14］ Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 9 rd ed. CLSI document M7-A9. Wayne， PA： Clinical and Laboratory Standards Institute； 2012.

［15］ Clinical and Laboratory Standards Institute. Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria. 2 rd ed. CSLI document M45-A2. Wayne， PA： Clinical and Laboratory Standards Institute； 2010.

［16］ Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing： Twenty-second information supplement，CLSI document M100-S22. Wayne， PA： Clinical and Laboratory Standards Institute； 2012.

［17］ Fahey JW. Moringa oleifera： A Review of the medical evidence for its nutricional， therapeutic， and prophylactic properties part 1. Trees Life J 2005； 1（5）： 1-15.

［18］ Onsare JG， Kaur H， Arora DS. Antimicrobial activity of Moringa oleifera from different locations against some human pathogens. Acad J Med Plant 2013； 1（5）： 80-91.

［19］ Austin B. Vibrios as casual agentes of zoonoses. Vet Microbiol 2010；140（3-4）： 310-317.

［20］ Igbinosa EO， Okoh AI. Emerging Vibrio species： an unending threat to public health in developing countries. Res Microbiol 2008； 159（7-8）： 495-506.

［21］ Ottaviani D， Leoni F， Rocchegiani E， Santarelli S， Masini L， Di Trani V，et al. Prevalence and virulence properties of non-O1， non-O139 Vibrio cholerae strains from seafood and clinical samples collected in Italy. Int J Food Microbiol 2009； 132（1）： 47-53.

［22］ Das BR， Kurup PA， Rao Narasimha PL. Antibiotic principle from Moringa pterygosperma. VII. Antibacterial activity and chemical structure of compounds related to pterygospermin. Indian J Med Res 1957； 45（2）：191-196.

［23］ Jabeen R， Shahid M， Jamil A， Ashraf M. Microscopic evaluation of the antimicrobial activity of seed extracts of Moringa oleifera. Pak J Bot 2008； 40（4）： 1349-1358.

［24］ Singh RSG， Negi PS， Radha， C. Phenolic composition， antioxidant and antimicrobial activities of free and bound phenolic extracts of Moringa oleifera seed flour. J Funct Foods 2013； 5（4）： 1883-1891.

R.S.N. Brilhante. Rua Coronel Nunes Melo， s/n， Rodolfo Teófilo. CEP： 60.430-270. Fortaleza， CE， Brazil.

Tel： 55 （85） 3366-8319

E-mail： brilhante@ufc.br

Foundation project： This work was supported by grants from the National Council for Scientific and Technological Development （CNPq； Brazil； Processes 445670/2014-2） and Coordination Office for the Improvement of Higher Education Personnel （AEI-0052-000650100/11）.