Quality of Probiotics for Food Animals

Thirteen commercial probiotic feed products were examined for microbiological content and the results were compared with the information available on the product labels. Antibiotic resistance of Lactobacillus and Bacillus was investigated. All the products were inaccurately labelled in either numbers or species of bacteria. Misnaming at the species level was the most common flaw. Lactobacillus exhibited higherantibiotic resistance than Bacillus did. Plasmid was found in both Lactobacillus (22%) and Bacillus (2.5%). The vanA gene was present in one L. plantarum and one B. subtilis isolate. The vanA-containing B. subtilis also harbored the tetW gene. None of the genes detected appeared to be associated with a conjugative plasmid. of probiotics depend on the number of viable bacteria, a minimal beneficial effect dose for probiotic bacteria has not been adequately established and most likely varies depending on the target animal and probiotic species. Most probiotic additives contain 1010 cfu/g and premixtures usually contain 108 cfu/g (Coeuret et al, 2004). However, several studies have reported that a number of commercial probiotic products contained low viable counts, resulting in a loss of probiotic effect (Hamilton-Miller et al, 1999; Temmerman et al, 2001; Coeuret et al, 2004). As safety and functionality of probiotics are species and strain dependent, recent reports have identified probiotic products with inaccurate species/strain labeling (Weese, 2003; Coeuret et al, 2004). These raise particular concerns regarding beneficial effects and potential health risks of the probiotic products. Lactobacillus and Bacillus have been formulated in several probiotic preparations sold commercially for veterinary use. Lactobacillus spp is an important part of the commensals of the animal body and has rarely INTRODUCTION Concerns that imprudent use of antibiotics in food animal production plays a role in widespread resistant of bacteria has been increasing. These resistant pathogens can be transmitted to humans through the food chain and contribute to a large pool of resistance genes that can be transferred to human pathogens. As a result, all antibiotic growth promoters have been banned and alternative feed ingredients, such as enzymes, organic acid, feed supplements and probiotics, have been researched and become commercially available. Together with the expanded market of organic farm animals, use of probiotics as animal feed additives has gained popularity. Probiotics are considered feed additives and subject to regulations depending the policy of each country. While health benefits SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 1104 Vol 40 No. 5 September 2009 been implicated in cases of infection. Bacillus spp is not an unusual component of microflora in gut and certain species are known to cause diseases: B. cereus may produce enterotoxin and B. pumilus has been associated with food poisoning and causes listeriosis like symptoms (Workowski and Flaherty, 1992). Recently, transmissible antibiotic resistance elements have been described in probiotic strains of Lactobacillus and Bacillus (Gevers et al, 2003; Hummel et al, 2007). A concern is these probiotic organisms may act as hosts for antibiotic resistance genes that are potentially transferred to commensal flora and pathogenic bacteria in the gut. In this case, widespread use of probiotic additives may result in increased distribution of potential sources for spread of antibiotic resistance genes. The aim of this study was to evaluate the quality of commercial probiotics used for animal consumption. The study was performed to enumerate and determine species of Lactobacillus and Bacillus. The accuracy of labeling on the products was assessed. Antimicrobial susceptibilities and horizontal transfer of antibiotic resistance genes were determined. MATERIALS AND METHODS Commercially available probiotic products Thirteen commercially available probiotics for food animals were evaluated in this study (Table 2). All products were freeze dried except product I which was in liquid form. Products II, III, IV, X, XI, XII and XIII were imported, the others were domestically manufactured. Five products, VII, IX, XI, XII and XIII, stated the expiration date on the product label and all of these products were tested at least 4 months before the expiration date. Two separate samples for each product were bought in bags or bottles. Each sample came from a different package. All products were stored at room temperature and analyzed within 7 days of being purchased. Isolation and enumeration of bacteria Isolation of Lactobacillus and Bacillus species was performed as described in ISO15214 and ISO7932, respectively. For dried products, a single 20 g portion from each sample was dissolved in 180 ml peptone saline diluting fluid (PSD; peptone 1.0 gm and NaCl 8.5 g in 1,000 ml distilled water). For liquid products, 1 ml of each product was diluted in 9 ml PSD. The samples were prepared in 10-fold dilutions and bacterial counts for each product were carried out in duplicate plates. The number of bacteria recorded for each sample was the mean of replicate counts. Counts of the total numbers of Lactobacillus and Bacillus were performed regardless of the species. Depending on the number of morphological types of colonies on an agar plate; 1-5 colonies of each type were randomly selected. All colonies were purified and subjected to Gram’s stain and biochemical testing. All bacterial isolates were stored as 20% glycerol stock at -80oC. Identification of genus and species Oligonucleotide primers used in this study are listed in Table 1. Genomic DNA was obtained from overnight cultures using QuickExtract (Epicentre® Biotechnologies, Madison, WI). Multiplex PCR assay was performed to verify genus and species of Lactobacillus (Nakagawa et al, 1994; Dubernet et al, 2002; Kwon et al, 2004). The Bacillus isolates were identified using amplified ribosomal DNA restriction analysis (ARDRA) (Wu et al, 2006). All PCR reactions were carried out using Eppendorf® MasterMix (Eppendorf, Hamburg, Germany) as described in the manufacturer’s instructions. The representatives of PCR products were submitted for nucleotide sequencing for conQUALITY OF PROBIOTICS FOR FOOD ANIMALS Vol 40 No. 5 September 2009 1105 Primers Sequence (5’-3’) PCR type Reference