An In Vitro Study on the Effect of some Pre and Probiotics on some pathogenic Bacteria

Potential strategies that could be incorporated with the current management practices include the use of probiotics (Poole et al., 2007). However, specific attention is also given to modern approaches for improving or stabilizing the intestinal system and its functioning by the deliberate application of viable microbial culture, so-called "probiotics", selected for special functional proberties (Holzapfel et al., 1998). Moreover, probiotics (specific microbial feeds with potential benefits to the host), and prebiotics (dietary components such as complex carbohydrates able to change the colonic microenvironment fostering colonization with non-enteropathogens) are areas of current interest because they offer alternatives for the management of the growing problem of multiple antibiotic resistance and overwhelming infections (Josephlevy, 1998). Since, natural agriculture or organic farming is a return for the nature or working with, but not against, the nature (Abdelhamid, 2002). So, the microbiota management tools of probiotics, prebiotics and synbiotics have been developed and, indeed, commercialized over the few decades with the expressed purpose of increasing number of bifidobacteria and/or lactobacilli within the gastrointestinal tract (Tuohy et al., 2003). Also, Castillo (2008) mentioned that among the numerous purported health benefits attributed to probiotic bacteria, their capacity to interact with the immune system of the host. New insights even show that certain Bacillus strains are able to block the communication system of enteropathogens. Therefore, the aim of the present work was to evaluate the effect of some commercial prebiotics and probiotics (in an in vitro study) on some pathogenic bacteria. 

The prebiotics "T-Protphyt 2000" and "Bio-Mos®":

T-Protphyt 2000 is a local product with a patent No. 23593. It consists of 15% zinc salts, 10% inorganic phosphorus, 5% dried fermentation products of Aspergillus oryzae growth, and starch as carrier up to one kilogram.  Each gram of this product contains 100 unit of phytase, 75 unit of protease, 25 unit of lipase, and 15 unit of amylase.

Bio-Mos® was gifted by the local marketing manager Vet. Med. Osamh El'Deep (Mobile: 002-0166606701). It is a natural product from Alltech®.For 15 years, Bio-Mos® has been revolutionizing animal feeding programs worldwide and is the only natural product for maintaining intestinal health supported by over 500 trials (peer-reviewed, university and practical field studies), solid market presence, quality assurance and technically sound expertise. This is why Bio-Mos® is a standard part of animal diets across the globe. Bio-Mos® is a yeast sugar derived from a select strain of Saccharomyces cerevisiae using a proprietary process developed by Alltech. Bio-Mos® is effective during all phases of growth. Bio-Mos® maintains intestinal health which is important for raising profitable animals. The intestinal tract is the largest organ in the body, giving rise to the largest surface area for the absorption and digestion of nutrients. Other functions of the intestinal tract include most active organ for defense system. Bio-Mos® is the brand of choice for helping animals achieve optimum performance. Everything starts with the development and maintenance of the intestinal tract. Bio-Mos® is useful for aquaculture. Bio-Mos® is Aggregate Rate Coefficient (ARC) certified as a quality control system from Alltech with every batch of Bio-Mos®. Its suggested rate for fish is 2-4 kg/ton (Proud sponsor of the Alltech FEI World Equestrian Games 2010^TM, www.alltech.com, and www.Bio-Moss.com).

Bacterial strains:

Nine of Gram-negative and positive bacteria, namely Aeromonas hydrophilla, Pseudomonas aeruginose, Pseudomonas fluorescens and Vibrio sp. in addition to the Enterobacteriaceae(Klebsiella, Shigella, Salmonella, Proteus, and Escherichia coli ) were used in this study, these  bacteria were isolated from Nile tilapia fish and identified by specific media and biochemical testes, except Aeromonas hydrophilla that was identified by primed polymerase chain reaction (PCR).

Assessment of antibacterial activity of the prebiotic:

Powder samples of pre-and probiotics and tetracycline (TC) antibiotic (because of its wide antibacterial spectrum and high potency, OCT is the most commonly used antibiotic against various diseases caused by Gram-negative and Gram-positive bacteria in fish farming) were suspended in sterile water and used at concentrations of (50 & 100 mg of either prebiotics and 30 mg of TC). Four wells were punched with a cork borer (6 mm in diameter) in plates of nutrient agar (NA) freshly seeded with 0.1 ml of 24 hr old of each tested bacterial cultures. Different concentrations of prebiotic and antibiotic were put into the wells, left one hr to allow diffusion; plates were incubated for 24hr at 37 °C. The diameter of clear zones surrounding the wells were measured and recorded expressing the antibacterial activity.

Three commercial products, namely two prebiotic (Superzyme Aqua and Garlen Extra 4, from Mediaavet, Kafer El-Sheikh) and one probiotic (Diamond V® original XP, from Egyvet, Mahala El-Koubra) from the local market were tested for their ability to act as bactericides. The 1^st is a product from Norel Co., Spain, it is a natural product composed of sodium butyrate, organic and non organic acids, zinc methionine, multi-enzyme complex with mainly endo-xylanase activity (from the fermentation of the fungus Trichoderma longibranchiatum). This prebiotic acts as bactericide on pathogenic germs but enhances the growth of beneficial bacteria. The 2^nd prebiotic  manufactured by Hefe Royal Eagle Co., China, is important for immunity health, containing 25% allicin, garlic extract. The 3^rd product the probiotic  Diamond V® original XP is a yeast (Saccharomyces cerevisiae) culture produced by Diamond V (Mills) Inc. Iowa, USA.

Six of Gram-negative bacteria namely Pseudomonas sp., Vibriosp., and Klebsiella (Gram-negative rods)  and  Micrococcus sp., Staphylococcus aureus, and Staphylococcus epidermidis ( Gram- positive cocci ) were used in this study, these bacteria were isolated from fish and identified by specific media and biochemical testes.

As in the 1^st case.

European Instand Dry Baker's Yeast is a Turkish product (Saccharomycescerevisiae) from Akmaya Sanayi Ve Ticaret A.S, imported by Al-Mansoura Co., Nasr City, Cairo. P ROBAX is an Indian yeast product. Whereas, Super Bio-Buds–2 X is a natural growth promoter consists of live cell yeast: 200 million cfu/g, calcium carbonate: 49.1%, corn distillers with soluble: 49.4%, and Saccharomycescerevisiae: 1.5%. It is a product from Brookside Agra, 2768 Troxler way,Highland,IL62249U.S.A. and imported by Tap Vet,Egypt. All these products were purchased from the local market.

As in the 2^nd case.

 Assessment of antibacterial activity of the probiotics:     

As in the 1^st and  2^nd cases. 

The diameter of clear zones surrounding the wells were measured and recorded expressing the antibacterial activity (Table 1 and Figs. 1 - 3).

Table 1: The antibacterial activity of aqueous prebiotics compared with TC.  

 

The diameter of clear zones surrounding the wells were measured and recorded expressing the antibacterial activity (Table 2 and Figs. 4 – 6).  

 

The diameter of clear zones surrounding the wells were measured and recorded expressing the antibacterial activity (Table 3 and Figs. 7 – 9).

Sahu et al. (2008) cited that aquaculture is one of the fastest developing growth sectors in the world andAsia presently contributes about 90% to the global production. However, disease outbreaks are constraint to aquaculture production thereby affects both economic development of the country and socio-economic status of the local people in many countries of Asia-Pacific region. Disease control in aquaculture industry has been achieved by following different methods using traditional ways, synthetic chemicals and antibiotics. However, the use of such expensive chemotherapeutants for controlling diseases has been widely criticized for their negative impacts like accumulation of residues, development of drug resistance, immunosuppressants and reduced consumer preference for aqua products treated with antibiotics and traditional methods are ineffective against controlling new diseases in large aquaculture systems. Therefore, alternative methods need to be developed to maintain a healthy microbial environment in the aquaculture systems there by to maintain the health of the cultured organisms. Use of probiotics is one of such method that is gaining importance in controlling potential pathogens.

Probiotics (specific microbial feeds with potential benefits to the host), and prebiotics (dietary components such as complex carbohydrates able to change the colonic microenvironment fostering colonization with non-enteropathogens) are areas of current interest because they offer alternatives for the management of the growing problem of multiple antibiotic resistance and overwhelming infections in the hospitalized patient (Josephlevy, 1998).

The research of probiotics for aquatic animals is increasing with the demand for environment friendly aquaculture. The probiotics were defined as live microbial feed supplements that biocontrol  and antagonist pathogens. Many other beneficial effects may be expected from probiotics, e.g., stimulation of the immune system. The most promising prospects are sketched out, but considerable efforts of research will be necessary to develop the applications to aquaculture (Gatesoupe, 1999 and Castillo, 2008).

Anyhow, Verschuere et al. (2000) reviewed the probiotic bacteria as biological control agents in aquaculture. They cited that in aquaculture system, the immediate ambient environment has a much larger influences on the health status than with terrestrial animals or humans; since in the aquatic environment, hosts and microorganisms share the ecosystem. Much more than terrestrial animals, aquatic farmed animals are surrounded by an environment that supports their pathogens independently of the host animals, and so (opportunistic) pathogens can reach high densities around the animal. Surrounding bacteria are continuously ingested either with the feed or when the host is drinking. They concluded that probiotics enhance the immune response, improve the water quality, and interact with phytoplankton. So, Poole et al. (2007) recorded that the emergence of multidrug resistant pathogens has stimulated a need to find alternatives to antimicrobials. Thus, Abdelhamid et al. (2008) used the Trichoderma viride for biological control of fungal (Saprolegnia sp. and Aspergillus ochraceus) diseases of fish.

Abd El-Rahman and El-Bana (2006) used Micrococcus luteus as a bacterial probiotic which presented in vitro and in vivo antagonistic effects against the pathogenic bacteria Aeromonas hydrophila. The inhibition zone to A. hydrophila was 40 mm in diameter due to M. luteus. Also, Taoka et al. (2006) indicated that probiotics treatment is promising as an alternative method to antibiotics for disease prevention in aquaculture. Moreover, Aly et al. (2008a) found that some Bacillus and Citrobacter strains isolated from Nile tilapia (B. pumilus, B. firmus, and C. freundii) showed inhibitory effects against A. hydrophila.  Also, Aly et al. (2008b) reported that the probiotic activity of two bacteria (Bacillus subtilis and Lactobacillusacidophilus) was evaluated by its effect on the immune response of Nile tilapia (Oreochromis niloticus), beside its protective effect against challenge infection.  The in-vitro antimicrobial assay showed that Bacillus subtilis and Lactobacillus acidophilus inhibited the growth of A. hydrophila. The B. subtilis inhibited the development of P. fluorescens while L. acidophilus inhibited the growth of Strept. iniae. The B. subtilis and L. acidophilus proved harmless when injected in the O. niloticus.  The serum bactericidal activity was high in the group that was given a mixture of the two bacteria. However, during the past two decades, the use of probiotics as an alternative to the use of antibiotics has shown to be promising in aquaculture, particularly in fish and shellfish larviculture (Tinh et al., 2008).

However, dietary supplementation of probiotic and vitamin C improved the immune response of fish (Nayak et al., 2007 and Panigrahi et al., 2007) as well as fish health (Panigrahi and Azad, 2007). Moreover, Abdel-Tawwab et al. (2008); Aly et al. (2008c); El-Ashram et al. (2008); Marzouk et al. (2008); Wang et al. (2008 a and b); Abd El-Aziz et al. (2009) and El-Nobi et al. (2009) studied the effect of probiotics on survival, resistance and quality of fish. Moreover, Abdelhamid et al. (2009) showed the positive effect of the prebiotic "T-Protphyt 2000" at the two concentrations against all the tested bathogenic bacteria and showed nearly no clear difference between the two concentrations of prebiotic. Also, it has a similar effect of that of the antibiotic (OTC), especially with the pathogenic bacteria, Aeromonas and Pseudomonas, which showed sensitivity towards prebiotic, while the Vibrio sp. showed resistance to OTC at the two concentrations. The prebiotics T-Protphyt 2000 and Bio-Mos^® (Abdelhamid et al., 2009 and 2012) improved also fish performance as a consecoence of the positive effects of the prebiotics on fish health and resistance.

Probiotic bacteria Micrococcusspecies isolated from the gonads of apparently healthy Oreochromis niloticus had antagonistic effect against the pathogenic Aeromonas hydrophilain vitro. The inhibition zone to A. hydrohpila was 47 mm in diameter due to M. species (Osman et al., 2010). Algedawy et al. (2011) concluded that the probiotic Biogen® is superior to the multienzyme mixture Natuzyme® for improving the cellular and humoral immune responses. Moreover, Dietary supplementation of B. subtilis at a dose of 1.35×10⁷ cfu g−1 improved growth, feed efficiency ratio, non-specific immune responses and disease resistance of juvenile large yellow croaker, L. crocea (Ai et al., 2011). 

Abd El-Aziz, M.; Ali, M. Yassin, S. Raafat, A. Moustafa, M. and Rashad, S. (2009). Effect of some probiotics on Oreochromis niloticus experimentally infected by Aeromonas hydrophila. Global Fisheries & Aquaculture Research Conference,Cairo, p: 24.

Abdelhamid, A.M. (2002). Organic Farming. Proc.2nd Conf. on Food borne Contamination and Egyptian’s Health, April 23 - 24, Mansoura Fac. Agric., pp: 69 – 77.

Abdelhamid, A.  M.; El-Barbary, M. I.  and Hasan, M. M. M. (2012). Effect of dietary supplementation with Bio-mos® or T-protphyt 2000 with and without hormone treatment on performance, chemical composition, and hormone residues of mono-sexNiletilapia. J. Animal and Poultry Production,MansouraUniversity, 3: 99-113.

Abdelhamid, A. M.; Mehrim, A. I.; El-Barbary, M. I.; Ibrahim, S. M.  and Abdel-Wahab, A. I. (2009). Evaluation of a new Egyptian probiotic by African catfish fingerlings. Journal of Environmental Science and Technology, 2 (2): 133 – 145 (engormix.com, Aquaculture Technical Articles, 13 p).

Abdelhamid, A. M., Shabana, Y. M. and Gomaa, S. S. A. (2008). Aquatic fungi and fish production in Egypt. I- In vitro studies. J. Agric. Sci. Mansoura Univ., 33: 4887-4899.

Abd El-Rahman, A.M.M. and El-Bana, L.F.A. (2006). Using of Micrococcus luteus as a probiotic bacteria among cultured Oreochromis niloticus, SCVMJ, X(1): 73 – 82.

Abdel-Tawwab, M., Abdel-Rahman, A.M and Ismael, N.E.M. (2008). Evaluation of commercial live bakers’ yeast, Saccharomyces cerevisiae as a growth and immunity promoter for Fry Nile tilapia, Oreochromis niloticus (L.) challenged in situ with Aeromonas hydrophila. Aquaculture, 280: 185–189.

Ai, Q.,  Xu, H.,  Mai, K.,  Xu, W.,  Wang, J. and Zhang, W. (2011). Effects of dietary supplementation of Bacillus subtilis and fructooligosaccharide on growth performance, survival, non-specific immune response and disease resistance of juvenile large yellow croaker, Larimichthys crocea. Aquaculture, 317: 155–161.

Algedawy, S. A., Soliman, K. K., Elashmawy, I.M. And El-Sayed, Y. S. (2011). Effect of commercial probiotic and exogenous enzymes on the growth performance, immune response, and hemato-biochemical parameters of broiler chickens. The 4^th Sci. Conf. of Animal Wealth Research in the Middle East & North Africa, p: 23 (Abstract).

Aly, S. M.; Abdel Atti, N. M. and Mohamed, M. F. (2008a). Effect of garlic on the survival, growth, resistance and quality of Oreochromis niloticus. 8th Inter. Symp. on Tilapia in Aquacul.,Cairo, pp: 277-296.

Aly, S.M., Abd-El-Rahman, A.M. John, G. and Mohamed, M.F. (2008b). Characterization of some bacteria isolated from Oreochromis niloticus and their potential use as probiotics. Aquaculture, 277: 1 – 6.

Aly, S.M., Ahmed, Y.A. Ghareeb, A.A. and Mohamed, M.F. (2008c). Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of tilapia nilotica (Oreochromis niloticus) to challenge infections. Fish and Shellfish Immunology, 25: 128 – 136.

Castillo, M. (2008). Blocking pathogens with Bacillus strains.  Feed-Mix, 16 (5): 19. (ISSN: 1754-5692).

El-Ashram, A.M.M., Mohammed, M.F. and Aly, S.M. (2008). Effect of Biobuds as a commercial probiotic product in cultured tilapia. 8^th International Symposium on Tilapia in Aquaculture, Oct.,Cairo,Egypt, pp: 1089 – 1096.

El-Nobi, G. A.; Reda, R. M. Hassanin, M. E. and Elmowalid, G. (2009). The impact of applications of probiotic Bacillus subtilis on health and growth of koi carp Cyprinus carpio. Global Fisheries & Aquaculture Research Conference,Cairo, p: 38.

Gatesoupe, F.J. (1999). The use of probiotics in aquaculture. Aquaculture, 180: 147–165.

Holzapfel, W.H., Haberer, P., Snel, J., Schillinger, U. and Veld, J. H. J. (1998). Overview of gut flora and probiotics. International Journal of Food Microbiology, 41: 85 – 101.                                                                        

Josephlevy, M. D. (1998). Immunonutrition: The Pediatric Experience. Nutrition, 14 (7/8): 641– 647.  

Marzouk, M.S., Moustafa, M.M.  and Mohamed, N.M.(2008). The influence of some probiotics on the growth performance and intestinal microbial flora of O. niloticus. 8^th International Symposium of Tilapia in Aquaculture, Oct.,Cairo,Egypt, pp: 1059 – 1071.

Nayak, S. K.; Swain, P. and Mukherjee, S. C. (2007). Effect of dietary supplementation of probiotic and vitamin C on the immune response of Indian major carp, Labeo rohita (Ham.). Fish & Shellfish Immunology, 23: 892-896.

Osman, H. A. M.; Ibrahim, T. B., Soliman, W. and Aboud, O. (2010). Improvement growth and immune status using a potential probiotic bacteria Micrococcus species among cultured Oreochromis niloticus.New York Science Journal, 3(10): 5-11.

Pangrahi, A. and Azad,I.S. (2007). Microbial intervention for better fish health in aquaculture: the Indian scenario. Fish Physiol. Biochem., 33: 429-440.

Pangrahi, A.; Kiron, V. Satoh, S. Hirono, I.Kobayashi, T. Sugita, H. Puangkaew, J. and Aoki, T. (2007). Immune modulation and expression of cytokine genes in rainbow trout Oncorhynchus mykiss upon probiotic feeding. Development and Comparative Immunology, 31: 372-382.

Poole, T., Callaway, T. and Nisbet, D. (2007). Alternatives to Antimicrobials. Infectious Disease: Foodborne Diseases. Edited by: S. Simjee © Humana Press Inc.,Totowa,NJ(p: 419).

Sahu, M.K.,Swarnakumar,N.S., Sivakumar, K., Thangaradjou, T. and Kannan, L. (2008). Probiotics in aquaculture: importance and future perspectives. Indian J. Microbiol., 10 p.

Taoka, Y., Meeda, H., Jo, T.Y., Kim, S.M., Park, S.I., Yoshikhawa, T.  and Sakata, T. (2006). Use of live and dead probiotic cells in tilapia Oreochromisniloticus. Fisheries Science, 72: 755 – 766.

Tinh, N.T.N., Direckens, K., Sorgeloos, P. and Bossier, P. (2008). A review of the functionality of probiotics in the larviculture food chain.  Marine Biotechnology, 10: 1 – 12.

Tuohy, K. M., Probert, H. M., Smejkal, C. W. and Gibson, G. R. (2003). Using  probiotics and prebiotics to improve gut health. DDD, 8: 692 – 700 (www.drugdiscoverytoday.com).

Verschuere, L.; Rombaut, G. Sorgeloos, P and Verstraete, W. (2000). Probiotic bacteria as biological control agents in aquaculture. Microbiology and Molecular Biology review, 64 (4): 655-671.

Wang, Y.; Li, J. and Lin, J. (2008a). Probiotics in aquaculture: Challenges and outlook. Aquaculture, 281: 1-4.

Wang, Y.B., Tian, Z.Q., Yao, J.T. and Li, W.F. (2008b). Effect of probiotics, Enteroccus faccium, on tilapia (Oreochromis niloticus) growth performance and immune response.  Aquaculture, 277: 203 – 207.