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November | December 2011

Feature title: Application of Enterococcus faecium as probiotic strain in aquatic species

The International magazine for the aquaculture feed industry

With the rapid developments in aquaculture fish and shrimp farming

industries are constantly under threat due to the outbreak of infec-tious diseases.

Members of the genus Vibrio and Aeromonas have been described as being among the most common pathogenic species in shrimp and fish causing seri-ous losses in larval and growout phases throughout the world. Use of antibiotics to control these agents has led to problems of drug resistance and resulted in trade restrictions in export markets.

As an alterna-tive strategy to these antimicro-bial compounds, the prophylactic use of beneficial bacteria (probiotics) has emerged to improve health and zootechni-cal performances such as survival, produc-

tion, feed conversion and growth rates of cultured aquatic species.

Probiotic bacteria are a sustainable approach to modulate the gut microflora towards a favorable composition, which is of utmost impor-

tance since the digestive system of fish and shrimp is an important entrance point for infections. Hence, selection criteria of probiotics for aquaculture should be based on their antagonism towards pathogens (through competi-tive exclusion), their growth, attachment to intestinal mucus and production of beneficial compounds (Vine et al., 2004).

Since fish and shrimp are cultured under different conditions the optimum temperature range should also be consid-ered for selection of the right probiotic

strain. Another important factor is that probiotics always lack pathogenicity.

After all, probiotics should contribute to efficient production in a sustainable way, promoting healthy and robust animals (Brittain et al., 2002).

Potential probiotic candidates

Lactic acid bacteria (LAB)

are potential probiotic candi-dates in aquaculture and are also known to be present in the intestine of healthy fish. Enterococcus faecium is one of the most commonly used lactic acid producing bacteria in animal nutrition and has become a focus of attention for use in commercially farmed aquatic species.

Enterococci are Gram-positive, faculta-tive anaerobic bacteria which are widely distributed in nature and considered as bacteria of low pathogenicity (Klare et al., 2003).

Probiotic enterococci infections have not been reported in the veterinary medicine, so the risk appears to be limited (Rinkinen et al., 2003). Eaton and Gasson (2001) found that E. faecium strains were also generally free of viru-lence determinants. Concerning possible acquired antibiotic resistances, testing the antibiotic profile of enterococci used as probiotics is of relevance.

However, these antibiotic resistances are species- or genus-specific properties (Klare et al., 2003). Once the probiotic enterococci are accurately identified, they can be tested for specific resistances.

Several important ways in which probi-

Picture 1: Microscopic picture (1000-fold magnification) of

Enterococcus faecium

Picture 2: Microscopic pictures (1000-fold magnification) of Vibrio harveyi, Yersinia ruckeri, Streptococcus agalactiae and Aeromons veronii

by Elisabeth Mayer MSc, Biomin, Austria

Application of Enterococcus faecium as probiotic strain in aquatic species

F: Enterococcus faecium

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otics producing lactic acid (such as E. fae-cium) can provide a performance benefit are improving intestinal microbial balance (Fuller, 1989), stimulating the immune system and decreasing pH as well as the release of bacteriocins (Rolfe, 2000).

Bacteriocins are small peptides which are characterized by their ability to inhibit pathogenic bacteria; whereas, some have a

narrow spectrum of activity while others inhibit a wide variety of bacteria.

The use of the bacteriocin-producing E. faecium with probiotic properties alone or in combination with other beneficial intestinal bacteria was investigated in different studies.

In vitro studies using the agar spot method (Rosskopf, 2010) have shown that Enterococcus faecium (strain IMB 52) has inhibition properties against a wide spectrum of aquatic pathogens including Yersinia ruckeri, Vibrio harveyi, Streptococcus agalactiae and Aeromonas veronii.

Similar observation was made by Swain et al. (2009) who proved the inhibi-tory activity of E. faecium isolated from brackishwater fish against V. harveyi and V. parahaemolyticus. This demonstrates the potential applications of E. faecium from fish intestine for controlling pathogenic vibriosis in shrimp culture. It has also been reported that survival rates of European eels (Anguilla Anguilla L.) fed with E. faecium were significantly higher than in the control groups after challenged with Edwardsiella tarda (Chang and Liu, 2002).

Wang et al. (2008) demonstrated that the addition of E. faecium (1 x 107 CFU/mL) in aquaria water could significantly increase final weight and daily weight gain (DWG) of tilapia. Since certain immunological param-eters (myeloperoxidase and respiratory burst activity) of tilapia were improved as well, the increased growth performance might be attributed to less bacterial chal-

lenge, confirming the benefit for the non-specific innate immunity of this kind of fish.

Panigrahi et al. (2007) examined immune modula-tion including cytokine gene expressions of rainbow trout (Oncorhynchus mykiss) and demonstrated that these parameters were improved by probiotic feeding of freeze-dried Lactobacillus rhamnosus, Enterococcus faecium or Bacillus subtilis (109 CFU/g)

after 45 days. Particularly the fish fed the E. faecium strain showed better perform-ance which could possibly be linked to the suitable ambient temperature condi-tions of this strain.

Temperature is a major environmental factor controlling microbial growth and the ideal conditions differ among micro-organisms. E. faecium was found to be more psychrotolerant than the other two bacteria, growing well at temperatures ranging from 12 to 30 °C. Rosskopf (2010) showed that the optimum tem-perature range for E. faecium (strain IMB 52) reaches as far as to 37 °C.

Using the fluorescence in situ hybridi-zation (FISH) technique, Supamattaya et al. (2005, 2006) have demonstrated in a series of in vivo studies that E. faecium (strain IMB 52) is also able to populate the intestine of white shrimp (Litopenaeus vannamei) and Nile tilapia (Oreochromis niloticus) (see Picture 3 and Table 1) and induce a positive impact on bacterial ecology of the gut by inhibiting Vibrio spp. through competitive exclusion.

E. faecium was even detected in the fish gut and faeces 10 days after product administration. In addition, it was observed that dietary application of E. faecium (strain IMB 52) alone or in combination can improve growth performance, enhance the immune response (by increasing granu-lar hemocytes level) (Supamattaya et al., 2005) and increase survival rate in shrimp contaminated with Vibrio parahaemolyticus (Krummenauer et al., 2009).

Means ± SD. Using Fluorescent in situ hybridization (FISH), 5 - 15 microscopic fields were counted.

From these studies it was concluded that Enterococcus faecium (strain IMB 52) is a safe and promising probiotic candidate for aquatic species which can be applied in different combinations.

ConclusionMaintaining the balance of critical

parameters and effective disease control remain fundamental requirements for suc-cessful aquaculture. In order to withstand the high stocking densities in shrimp and fish production probiotics are a promising feed additive to stimulate animal growth and advance disease resistance.

Enterococcus faecium as probiotic strain in aquatic species is increasingly recognized as safe and can be applied in different combinations. E. faecium can grow at a wide range of temperatures and thus has an advantage over other bacteria considering that fish and shrimp are reared at different temperatures and conditions.

The current paper illustrated with in vitro studies and performance trials the potential benefits of E. faecium as probiotic feed additive for fish and shrimp, either by stimulating the development of a healthy gut microflora or by inhibit-ing pathogenic bacteria like Vibrio spp., Yersinia spp. and Aeromonas spp.

ReferencesAvailable on request

Table 1: E. faecium in tilapia´s intestine and faeces at day one and day 10 after stopping to feed probiotics including this probiotic strain

Experimental group E. faecium in tilapia´s intestine (x 108 cell/g intestine)

Day 1 Day 10

: Intestine with faecal materials : Faecal material : Intestine w/o faeces

Control group 0 0 0

Probiotic group 1.37 ± 0.85 1.52 ± 0.98 1.33 ± 0.28

14 | InternatIonal AquAFeed | november-December 2011 november-December 2011 | InternatIonal AquAFeed | 15

F: Enterococcus faecium

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