Comparison and Utilization of Repetitive-Element PCR Techniques for Typing Lactobacillus Isolates from the Chicken Gastrointestinal Tract

Abstract

Three repetitive-element PCR techniques were evaluated for the ability to type strains of Lactobacillus species commonly identified in the chicken gastrointestinal tract. Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) produced species-and strain-specific profiles for Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri isolates. The technique typed strains within these species equally as well as pulsed-field gel electrophoresis. DNA concentration and quality did not affect the ERIC-PCR profiles, indicating that this method, unlike other high-resolution methods, can be adapted to high-throughput analysis of isolates. Subsequently, ERIC-PCR was used to type Lactobacillus species diversity of a large collection of isolates derived from chickens grown under commercial and necrotic enteritis disease induction conditions. This study has illustrated, for the first time, that there is great strain diversity within each Lactobacillus species present and has revealed that chickens raised under commercial conditions harbor greater species and strain diversity than chickens raised under necrotic enteritis disease induction conditions.

Lactobacillus Strain Ecology and Persistence within Broiler Chickens Fed Different Diets: Identification of Persistent Strains

“…All LAB cultures were grown in MRS broth (Difco, Detroit, MI) or agar at 37°C for 24 or 48 h, respectively, under anaerobic conditions using a BD GasPAK EZ container (Becton Dickinson, Sydney, Australia) with an AnaeroGen sachet (Oxoid Australia, Adelaide, Australia). The strains used in this study were collected previously (48), and the species distribution of the strain mixes used in this study are listed in Table 1.…”

“…The "selected strains" specified in Table 1 were chosen from commercial and NE chicken isolates identified previously (48) and were chosen based upon multiple isolations from at least two chickens and from two or more different regions of the GIT. Strain mixes 1 and 2 were comprised of random collections of strains recovered from chickens fed commercial and high-protein diets.…”

“…One of the limitations of in vivo screening of lactobacilli is the need for reliable high-throughput screening techniques to identify and track persistent strains. Recently, our group reported the application of enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) to simultaneously type large numbers of Lacto-bacillus isolates from the chicken GIT to the species and strain level (48).…”

Transformation of, and Heterologous Protein Expression in, Lactobacillus agilis and Lactobacillus vaginalis Isolates from the Chicken Gastrointestinal Tract

“…Bacterial strains used in this study are listed in Table 1. Lactobacillus agilis La3 and L. crispatus Lc9 were originally isolated from the jejuna and L. vaginalis Lv5 was isolated from the crops of chickens fed an experimental high-protein diet (44) and subsequently found to persist in the chicken GIT upon reinoculation (45). Lactobacilli were routinely cultured in MRS broth (Difco, Detroit, MI) and agar at 37°C for 24 and 48 h, respectively, under anaerobic conditions using a BD GasPAK EZ container (Becton Dickinson, Sydney, Australia) with an AnaeroGen sachet (Oxoid Australia, Adelaide, Australia).…”

“…DNA isolation and manipulations. Genomic DNA from Lactobacillus strains was isolated using the method described previously (44). Plasmid DNA was isolated from E. coli using the Qiagen plasmid midikit (Qiagen Pty Ltd., Melbourne, Australia) for large-scale preparations, while small-scale preparations were performed using the Wizard Plus SV minipreps DNA purification system (Promega, Sydney, Australia).…”