R E S E A R C H L E T T E R Estimation of 16S rRNA gene copy number in several probiotic Lactobacillus strains isolated from the gastrointestinal tract of chicken Chin Mei Lee1, Chin Chin Sieo1,2, Norhani Abdullah1,2 & Yin Wan Ho1 1Microbial Biotechnology Unit, Laboratory of Industrial Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia; and 2Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Selangor, Malaysia OnlineOpen: This article is available free online at www.blackwell-synergy.com Correspondence: Chin Chin Sieo, Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: 1603 8946 6702; fax: 1603 8943 0913; e-mail: ccsieo@biotech.upm.edu.my Received 27 December 2007; accepted 16 July 2008. First published online 14 August 2008. DOI:10.1111/j.1574-6968.2008.01305.x Editor: Wolfgang Kneifel Keywords Lactobacillus spp.; 16S rRNA gene copy number; rRNA restriction patterns. Abstract The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe comple- mentary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 was estimated to have seven copies of the 16S rRNA gene, Lactobacillus panis C 17 to have ﬁve copies and Lactobacillus gallinarum strains I 16 and I 26 four copies. The 16S rRNA gene copy numbers of L. gallinarum and L. panis reported in the present study are the ﬁrst record. Lactobacillus brevis strains I 12, I 23, I 25, I 211, I 218 and Lactobacillus reuteri strains C 1, C 10, C 16 were estimated to have at least four copies of the 16S rRNA gene. In addition, distinct rRNA restriction patterns which could discriminate the strains of L. reuteri and L. gallinarum were also detected. Information on 16S rRNA gene copy number is important for physiological, evolutionary and population studies of the bacteria. Introduction Lactobacilli, the predominant species in the avian alimen- tary tract, are Gram-positive, nonsporing, catalase-negative, devoid of cytochromes and acid tolerant bacteria. They are strictly fermentative, with lactic acid as the major end- product during carbohydrate fermentation. The concept of using lactobacilli as probiotics to exert health beneﬁts has drawn the attention of the public in recent years, particu- larly following the total ban or severe restriction of anti- biotics as growth promoters in the animal industry. With the growing interest in the use of probiotics, it is essential to have a rapid and accurate method for assessing probiotic preparations. Various molecular techniques, which are known to be rapid, accurate and sensitive, could be em- ployed for this purpose. The most widely used techniques for bacterial identiﬁcation and quantiﬁcation are based on the 16S rRNA gene. Previous studies have indicated that the c 2008 The Authors Journal compilation c 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. 16S rRNA gene is present in multiple copies in most bacterial genomes and the copy numbers vary from one to as many as 15 (Acinas et al., 2004). The 16S rRNA gene copy number is useful for 16S rRNA gene-based quantiﬁcation technique. Data obtained from samples for the estimation of microbial community should be normalized with the 16S rRNA gene copy number per cell to avoid quantitative bias. To date, Lactobacillus 16S rRNA gene copy number has only been characterized in a few species. Lactobacillus acidophilus has been reported to comprise four copies of the 16S rRNA gene (Roussel et al., 1993; Altermann et al., 2005). Lactobacillus plantarum (Chevallier et al., 1994; Kleerebezem et al., 2003) and Lactobacillus brevis (Makarova et al., 2006) contain ﬁve copies. Six copies of the 16S rRNA gene are found in the genome of Lactobacillus johnsonii (Pridmore et al., 2004), Lactobacillus gaseri (Makarova et al., 2006) and Lactobacillus reuteri (GenBank accession number CP000705). Lactobacillus sakei (Dudez et al., 2002) and FEMS Microbiol Lett 287 (2008) 136–141