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The effect of Quercus castaneifolia extract on pathogenic enteric bacteria
Anaerobe 17 (2011) 358e360
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Pathogenesis and Toxins
The effect of Quercus castaneifolia extract on pathogenic enteric bacteria N. Bahador a, *, M. Baserisalehi b a b
Department of Microbiology, Science and Research Branch, Islamic Azad University, Fars, Iran Department of Microbiology, Kazeroun Branch, Islamic Azad University, Kazeroun, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 January 2011 Received in revised form 14 July 2011 Accepted 16 July 2011 Available online 2 August 2011
The family of Enterobacteriaceae is a major group of gram negative bacteria, some of these microorganisms are pathogen and could cause disease mainly gastroenteritis. Recently, due to drug resistant nature of these bacteria specially in developing countries treatment of the patient considered as important investigate. Quercus castaneifolia is a native plant of Yasuj province in Iran, which the people who living in this area consume the fruit of this plant for treatment of enteric disease. Hence, the present study was conducted to evaluate the effect of fruit of Q. castaneifolia extract on pathogenic enteric bacteria viz., E. coli, Salmonella typhimurium, Shigella dysenteriae and Yersinia enterocolitica. Antimicrobial susceptibility and minimal inhibitory concentration (MIC) of the extracts were assessed by gel diffusion method and modification of E-test respectively. All the experiments were performed in triplicate and the statistical analysis was carried out on the results. The results obtained from this study indicated that alcoholic extract was shown antimicrobial effect on the microorganisms tested. In addition, S. dysentriae was more sensitive with zone of inhibition 18 mm and MIC value was 2.5 10 4 whereas, E. coli was less sensitive with zone of inhibition 12 mm and MIC value 1 10 2. Salmonella typhimurium and Yersinia enterocolitica showed relatively intermediate susceptibility to the extract with zone of inhibition of 14 mm and MIC value 5 10 3. Overall, Q. castaneifolia may be considered for treatment of the patients suffering from enteric disease. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Enteric bacteria Quercus castaneifolia Antimicrobial susceptibility
1. Introduction The family of Enterobacteriacea is a major group of gram negative bacteria, which living in soil, water, human and animal bodies. Based on 16s RNA sequencing the family belongs to the g proteobacteria. Characteristics of these bacteria are cocco bacilli, facultative anaerobic, non spore forming, catalase positive with negative oxidase production. They ferment sugar and produce lactic acid and most of them reduce nitrate. Due to existence of flagella most of them are motile but a few genera are non-motile. In addition, presence of peritrichous Type I fimbriae in this family involved in the adhesion of the bacterial cells to their hosts and cause disease. On the other hand, some of these microorganisms are pathogen and could cause disease mainly gastroenteritis. Escherichia coli, is one of the most important organism in this family which have been closely studied. Furthermore, Klebsiella and Proteus sometimes cause urinary tract infections; and pneumonia occurs generally in immunocompromised hosts or alcoholics [4]. In
* Corresponding author. E-mail address: [email protected] (N. Bahador). 1075-9964/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2011.07.007
addition, Salmonella is closely related to the genus of Escherichia and are found worldwide in cold- and warm-blooded animals (including humans), and in the environment. They cause illnesses such as typhoid fever, paratyphoid fever and food borne illness [13]. The shell of eggs may be contaminated with Salmonella by feces or environment, or the bacterium can infect the eggs via infected ovaries [5]. Therefore, evaluation of the eggs or other samples can help us to control the disease. Over the past 20 years exploration of natural materials as source of new antibacterial agent without side effect has been started. For this purpose, different extracts from traditional medicinal plants have been tested to identify the source of therapeutic effect [8]. Hence, herbal medicine represents one of the most important fields of traditional medicine all over the world. On the other hand [6], believed that in order to use of herbal medicines and determination of their potential as a source of new drug it is necessary to study medicinal plants. Recently, due to multi drug resistance of bacteria, usage of plant and other natural material extract is in progress. Therefore, the present study conducted to evaluate fruit extract of Quercus castaneifolia on the pathogenic enteric bacteria in order to achieve the best manner for solving the problem of enteric bacterial disease regarding to antibiotic resistance.
N. Bahador, M. Baserisalehi / Anaerobe 17 (2011) 358e360
359
Table 1 Antimicrobial susceptibility of fruit of Quercus castaneifolia extract. Microorganisms
Dilutions 1/2
1/10
Zone of inhibition (mm) Escherichia coli Shigella dysentriae Salmonella typhimurium Yersinia enterocolitica
12 18 14 14
11.6 14.6 12.6 12
The data point is average of three replicates.
Table 2 Minimum inhibitory concentration for the bacteria.
Fig. 1. Effect of the alcoholic extract on Shigella dysentriae.
2. Material and methods
Microorganisms
MIC Value (mg/l)
Escherichia coli Shigella dysentriae Salmonella typhimurium Yersinia enterocolitica
1 10 2 2.5 10 5 10 3 5 10 3
4
The data point is average of three replicates.
The fruit of Q. castaneifolia has been collected from Yasuj province, Iran. The samples were washed under tapping water, dried under shade and powdered. To prepare water extract of the fruit, 50 g of air dried fine powder was placed into the flask with 100 ml hot water, and for preparation of alcoholic extract the described procedure was repeated except alcohol was used in place of hot water. The flasks were plugged with cotton and then kept on rotary shaker for 3 days. Then the contents were centrifuged at 2500 rpm for 15 min. The supernatant was collected and the solvent was evaporated to make higher concentration and it was stored at 4 C in airtight bottles for further studies [8]. 2.1. Test organisms The test organisms used in this study were E. coli (PTCC1533), Salmonella thyphimurium (PTCC1230), Shigella dysentriae (PTCC1188), Yersinia enterocolitica (PTCC1477). 2.2. Antimicrobial activity (gel diffusion method, modified E
test)
A loop full standard strains of E. coli, S. thyphimurium, S. dysentriae, and Yersinia enteroclitica were inoculated into 30 ml nutrient broth in a conical flasks. The flasks were incubated for 24 h at 37 C to activate the strains. Antimicrobial susceptibility and
minimal inhibitory concentration (MIC) of the extracts were assessed by gel diffusion method and modified E-test respectively [2]. For this purpose 0.1 ml of the inoculums were poured on the medium and spread properly. The extracted compounds were introduced into the well and the plates were incubated at 37 C for 24 h. All samples were tested in triplicates and the results were recorded. 3. Results and discussion The results obtained from this study indicated that alcoholic extract has shown antimicrobial effect on the microorganisms tested (Figs. 1 and 2). While the aqueous extract has not affect on the same organisms. In addition, S. dysentriae was more sensitive with zone of inhibition 18 mm and MIC value was 2.5 10 4 whereas, E. coli was less sensitive with zone of inhibition 12 mm and MIC value 1 10 2. In addition, S. typhimurium and Y. entrocolitica showed relatively intermediate susceptibility to the extract with zone of inhibition 14 mm and MIC value 5 10 3 (Tables 1 and 2). In last years many scientists have been worked on antimicrobial activity of flowers against dominant and pathogenic bacteria [1,3,9,11]. In 2006 Nayak and Pereira [12] observed that Catharanthus roseus flower extract have wound healing activity in rats and in 2008 Mehlika et al. [10], showed that leaf and flower of six endemic plant species had antimicrobial activity against Staphylococcus aureus and Bacillus subtilis. In addition [7] studied on different part of plants (roots, stems, leaves, flowers and fruits) for their antimicrobial activity against pathogenic bacteria and all of the scientists believe that the natural materials or plant extracts has affect on the pathogenic bacteria. Although, enteric bacteria can cause enteritis in human being, our investigates illustrated that the extracts has antimicrobial activity on the organisms tested hence, consumption of these fruits could reduce the infection which rise by the same bacteria. On the other hand, due to increasing rate of antibiotic resistant enteric bacteria, treatment of the enteritis could be done by herbal medicine such as Q. castanifolia. References
Fig. 2. Effect of the alcoholic extract on Escherichia coli.
[1] Ahmed L, Mohammed Z, Mohammed F. Screening of some Indian medicational plants for their antimicrobial properties. J Ethanopharmacol 1988;62: 183e93.
360
N. Bahador, M. Baserisalehi / Anaerobe 17 (2011) 358e360
[2] Baserisalehi M, Al. Mahdi AY, Kapadnis BP. Antimicrobial susceptibility of thermophilic Campylobacter spp. isolated from environmental samples. Indian J Med Microbiol 2005;23:48e51. [3] Benson HJ. In: Microbiological applications. 5th ed. USA: WMC. Brwon Publ.; 1990. p. 134. Cragg, G.M., Newman, D.J. and Snader, K.M.1997.Natural products in Prod., 60: 52e60. [4] http://www.Healthofchildren.com [5] Humphrey TJ. Contamination of egg shell and contents with Salmonella enteritidis: a review. Int J Microbiol 1994;21(1e2):31e40. [6] Jigna P, Sumitra C. In vitro antibacterial activity of the crude methanol extract of Woodfordia fruticosa flowe. Brazilian J Microbiol 2007;38:204e7. [7] Juliana B, Valdir C, Vania FN, Mara RK, Daniela EB, Alexandre BC. Antimicrobial activity of fractions and compounds from Calophyllum brasiliense. Naturforsch 2004;59:657e62.
[8] Kandukuri Vasu, Singara Charya. Antimicrobial of five flowers extracts aginst three pathogenic bacteria. J Nat Environ Pollut Technol 2010; 9(1):73e6. [9] Kudi AC, Umoh JU, Eduvie LO, Gefu J. Screening of some Nigerian medicinal plants for antibacterial activity. J Ethanopharmacol 1999;67:225e8. [10] Mehlika B, Umit B, Fatmagul G, Kerim G, Nazife Y. An investigation on the antimicrobial activity of some endemic plant species from Turkey. Afr J Biotech 2008;7:1e5. [11] Nair R, Kalariya T, Chanda S. Antibacterial activity of some selected Indian medicinal flora. Turk J Biol 2005;29:41e7. [12] Nayak BS, Pereira MP. Catharanthus roseus flower extract has wound-healing activity in Sprague Dawley rats. BMC Alt Med 2006;6:41. [13] Ryan KJ, Ray CG, editors. Sherris medical microbiology. 4th ed. McGraw Hill; 2004. p. 362e8.
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Pathogenesis and Toxins
The effect of Quercus castaneifolia extract on pathogenic enteric bacteria N. Bahador a, *, M. Baserisalehi b a b
Department of Microbiology, Science and Research Branch, Islamic Azad University, Fars, Iran Department of Microbiology, Kazeroun Branch, Islamic Azad University, Kazeroun, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 January 2011 Received in revised form 14 July 2011 Accepted 16 July 2011 Available online 2 August 2011
The family of Enterobacteriaceae is a major group of gram negative bacteria, some of these microorganisms are pathogen and could cause disease mainly gastroenteritis. Recently, due to drug resistant nature of these bacteria specially in developing countries treatment of the patient considered as important investigate. Quercus castaneifolia is a native plant of Yasuj province in Iran, which the people who living in this area consume the fruit of this plant for treatment of enteric disease. Hence, the present study was conducted to evaluate the effect of fruit of Q. castaneifolia extract on pathogenic enteric bacteria viz., E. coli, Salmonella typhimurium, Shigella dysenteriae and Yersinia enterocolitica. Antimicrobial susceptibility and minimal inhibitory concentration (MIC) of the extracts were assessed by gel diffusion method and modification of E-test respectively. All the experiments were performed in triplicate and the statistical analysis was carried out on the results. The results obtained from this study indicated that alcoholic extract was shown antimicrobial effect on the microorganisms tested. In addition, S. dysentriae was more sensitive with zone of inhibition 18 mm and MIC value was 2.5 10 4 whereas, E. coli was less sensitive with zone of inhibition 12 mm and MIC value 1 10 2. Salmonella typhimurium and Yersinia enterocolitica showed relatively intermediate susceptibility to the extract with zone of inhibition of 14 mm and MIC value 5 10 3. Overall, Q. castaneifolia may be considered for treatment of the patients suffering from enteric disease. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Enteric bacteria Quercus castaneifolia Antimicrobial susceptibility
1. Introduction The family of Enterobacteriacea is a major group of gram negative bacteria, which living in soil, water, human and animal bodies. Based on 16s RNA sequencing the family belongs to the g proteobacteria. Characteristics of these bacteria are cocco bacilli, facultative anaerobic, non spore forming, catalase positive with negative oxidase production. They ferment sugar and produce lactic acid and most of them reduce nitrate. Due to existence of flagella most of them are motile but a few genera are non-motile. In addition, presence of peritrichous Type I fimbriae in this family involved in the adhesion of the bacterial cells to their hosts and cause disease. On the other hand, some of these microorganisms are pathogen and could cause disease mainly gastroenteritis. Escherichia coli, is one of the most important organism in this family which have been closely studied. Furthermore, Klebsiella and Proteus sometimes cause urinary tract infections; and pneumonia occurs generally in immunocompromised hosts or alcoholics [4]. In
* Corresponding author. E-mail address: [email protected] (N. Bahador). 1075-9964/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2011.07.007
addition, Salmonella is closely related to the genus of Escherichia and are found worldwide in cold- and warm-blooded animals (including humans), and in the environment. They cause illnesses such as typhoid fever, paratyphoid fever and food borne illness [13]. The shell of eggs may be contaminated with Salmonella by feces or environment, or the bacterium can infect the eggs via infected ovaries [5]. Therefore, evaluation of the eggs or other samples can help us to control the disease. Over the past 20 years exploration of natural materials as source of new antibacterial agent without side effect has been started. For this purpose, different extracts from traditional medicinal plants have been tested to identify the source of therapeutic effect [8]. Hence, herbal medicine represents one of the most important fields of traditional medicine all over the world. On the other hand [6], believed that in order to use of herbal medicines and determination of their potential as a source of new drug it is necessary to study medicinal plants. Recently, due to multi drug resistance of bacteria, usage of plant and other natural material extract is in progress. Therefore, the present study conducted to evaluate fruit extract of Quercus castaneifolia on the pathogenic enteric bacteria in order to achieve the best manner for solving the problem of enteric bacterial disease regarding to antibiotic resistance.
N. Bahador, M. Baserisalehi / Anaerobe 17 (2011) 358e360
359
Table 1 Antimicrobial susceptibility of fruit of Quercus castaneifolia extract. Microorganisms
Dilutions 1/2
1/10
Zone of inhibition (mm) Escherichia coli Shigella dysentriae Salmonella typhimurium Yersinia enterocolitica
12 18 14 14
11.6 14.6 12.6 12
The data point is average of three replicates.
Table 2 Minimum inhibitory concentration for the bacteria.
Fig. 1. Effect of the alcoholic extract on Shigella dysentriae.
2. Material and methods
Microorganisms
MIC Value (mg/l)
Escherichia coli Shigella dysentriae Salmonella typhimurium Yersinia enterocolitica
1 10 2 2.5 10 5 10 3 5 10 3
4
The data point is average of three replicates.
The fruit of Q. castaneifolia has been collected from Yasuj province, Iran. The samples were washed under tapping water, dried under shade and powdered. To prepare water extract of the fruit, 50 g of air dried fine powder was placed into the flask with 100 ml hot water, and for preparation of alcoholic extract the described procedure was repeated except alcohol was used in place of hot water. The flasks were plugged with cotton and then kept on rotary shaker for 3 days. Then the contents were centrifuged at 2500 rpm for 15 min. The supernatant was collected and the solvent was evaporated to make higher concentration and it was stored at 4 C in airtight bottles for further studies [8]. 2.1. Test organisms The test organisms used in this study were E. coli (PTCC1533), Salmonella thyphimurium (PTCC1230), Shigella dysentriae (PTCC1188), Yersinia enterocolitica (PTCC1477). 2.2. Antimicrobial activity (gel diffusion method, modified E
test)
A loop full standard strains of E. coli, S. thyphimurium, S. dysentriae, and Yersinia enteroclitica were inoculated into 30 ml nutrient broth in a conical flasks. The flasks were incubated for 24 h at 37 C to activate the strains. Antimicrobial susceptibility and
minimal inhibitory concentration (MIC) of the extracts were assessed by gel diffusion method and modified E-test respectively [2]. For this purpose 0.1 ml of the inoculums were poured on the medium and spread properly. The extracted compounds were introduced into the well and the plates were incubated at 37 C for 24 h. All samples were tested in triplicates and the results were recorded. 3. Results and discussion The results obtained from this study indicated that alcoholic extract has shown antimicrobial effect on the microorganisms tested (Figs. 1 and 2). While the aqueous extract has not affect on the same organisms. In addition, S. dysentriae was more sensitive with zone of inhibition 18 mm and MIC value was 2.5 10 4 whereas, E. coli was less sensitive with zone of inhibition 12 mm and MIC value 1 10 2. In addition, S. typhimurium and Y. entrocolitica showed relatively intermediate susceptibility to the extract with zone of inhibition 14 mm and MIC value 5 10 3 (Tables 1 and 2). In last years many scientists have been worked on antimicrobial activity of flowers against dominant and pathogenic bacteria [1,3,9,11]. In 2006 Nayak and Pereira [12] observed that Catharanthus roseus flower extract have wound healing activity in rats and in 2008 Mehlika et al. [10], showed that leaf and flower of six endemic plant species had antimicrobial activity against Staphylococcus aureus and Bacillus subtilis. In addition [7] studied on different part of plants (roots, stems, leaves, flowers and fruits) for their antimicrobial activity against pathogenic bacteria and all of the scientists believe that the natural materials or plant extracts has affect on the pathogenic bacteria. Although, enteric bacteria can cause enteritis in human being, our investigates illustrated that the extracts has antimicrobial activity on the organisms tested hence, consumption of these fruits could reduce the infection which rise by the same bacteria. On the other hand, due to increasing rate of antibiotic resistant enteric bacteria, treatment of the enteritis could be done by herbal medicine such as Q. castanifolia. References
Fig. 2. Effect of the alcoholic extract on Escherichia coli.
[1] Ahmed L, Mohammed Z, Mohammed F. Screening of some Indian medicational plants for their antimicrobial properties. J Ethanopharmacol 1988;62: 183e93.
360
N. Bahador, M. Baserisalehi / Anaerobe 17 (2011) 358e360
[2] Baserisalehi M, Al. Mahdi AY, Kapadnis BP. Antimicrobial susceptibility of thermophilic Campylobacter spp. isolated from environmental samples. Indian J Med Microbiol 2005;23:48e51. [3] Benson HJ. In: Microbiological applications. 5th ed. USA: WMC. Brwon Publ.; 1990. p. 134. Cragg, G.M., Newman, D.J. and Snader, K.M.1997.Natural products in Prod., 60: 52e60. [4] http://www.Healthofchildren.com [5] Humphrey TJ. Contamination of egg shell and contents with Salmonella enteritidis: a review. Int J Microbiol 1994;21(1e2):31e40. [6] Jigna P, Sumitra C. In vitro antibacterial activity of the crude methanol extract of Woodfordia fruticosa flowe. Brazilian J Microbiol 2007;38:204e7. [7] Juliana B, Valdir C, Vania FN, Mara RK, Daniela EB, Alexandre BC. Antimicrobial activity of fractions and compounds from Calophyllum brasiliense. Naturforsch 2004;59:657e62.
[8] Kandukuri Vasu, Singara Charya. Antimicrobial of five flowers extracts aginst three pathogenic bacteria. J Nat Environ Pollut Technol 2010; 9(1):73e6. [9] Kudi AC, Umoh JU, Eduvie LO, Gefu J. Screening of some Nigerian medicinal plants for antibacterial activity. J Ethanopharmacol 1999;67:225e8. [10] Mehlika B, Umit B, Fatmagul G, Kerim G, Nazife Y. An investigation on the antimicrobial activity of some endemic plant species from Turkey. Afr J Biotech 2008;7:1e5. [11] Nair R, Kalariya T, Chanda S. Antibacterial activity of some selected Indian medicinal flora. Turk J Biol 2005;29:41e7. [12] Nayak BS, Pereira MP. Catharanthus roseus flower extract has wound-healing activity in Sprague Dawley rats. BMC Alt Med 2006;6:41. [13] Ryan KJ, Ray CG, editors. Sherris medical microbiology. 4th ed. McGraw Hill; 2004. p. 362e8.