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

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.Lactobacilli are normal inhabitants within the microflora of the chicken gastrointestinal tract (GIT) (27, 39). Species frequently identified within the chicken GIT include Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri (1, 7, 27). The first three of these species are members of the Lactobacillus acidophilus complex (LAC) (22, 32), a closely related group of species which are difficult to differentiate using traditional techniques, such as physiological and biochemical tests (34). While molecular methods, such as DNA-DNA hybridization (22), ribotyping (71), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (19), randomly amplified polymorphic DNA PCR (19), and 16S rRNA gene sequencing (41), have been used with some success, many of these techniques are not readily adaptable to high-throughput applications required for large-scale ecological studies.While numerous studies have reported Lactobacillus species distribution within the chicken GIT, the strain diversity within species has not been explored. Previously, Hagen et al. (28) investigated L. gallinarum isolates present within the crops of commercial chickens, revealing a high level of strain diversity among the isolates examined (17 strains represented among 38 isolates). These results indicate that there could be great diversity within and among the lactobacilli present within the chicken GIT. Examining and typing large numbers of lactobacilli from the chicken GIT to the strain level may facilitate a better understanding of microflora dynamics and niche competition. These studies may also result in the identification of strains which could be used in competitive exclusion applications and potentially in the development of probiotics or live vectors for the delivery of therapeutic recombinant proteins to specific sites within the chicken GIT.Lactobacilli have been proposed as possible competitive exclusion agents or probiotics (30, 36, 42) against Clostridium perfringens, the causative agent of necrotic enteritis (NE) in broiler chickens. The withdrawal of antimicrobial growth promoters in Europe has led to an increase in the incidence of NE (10, 64), prompting the investigation of alternative methods for controlling C. perfringens in the broiler chicken GIT. Various models have been developed to study NE under research conditions, as recently reviewed by Dahiya et al. (15). The conditions used in these NE models and the effects they have on GIT microflora may adversely impact the identification of antimicrobial alternatives, such as probiotic strains, for use within commercial broiler chickens. Application of a high-throughput typing method capable of distinguishing species and strains is needed to determine if differences exist between the Lactobacillus populations of chickens raised under NE and commercial conditions.While pulsed-field gel electrophoresis (PFGE) has been used widely for genotyping Lactobacillus strains (49, 50, 65), it is time-consuming, labor-intensive, expensive, and suitable only for low-throughput analysis of isolates (54, 65). In contrast, repetitive-element PCR (Rep-PCR) has been developed for genotypically fingerprinting bacteria and is a fast and reliable high-throughput genotyping system (66, 67). Rep-PCR has been used successfully to identify strains of a variety of genera (33, 44, 48, 55). Several Rep-PCR primers, including the repetitive extragenic palindromic (REP) primers (6, 9, 16, 65), the enterobacterial repetitive intergenic consensus (ERIC) primers (6, 65), and the (GTG)₅ primer (24, 40, 62), have been used in the typing of lactobacilli in studies which have generally focused on species important to the dairy industry and food fermentations. Very few studies have examined the application of Rep-PCR to species commonly identified in the chicken GIT (62, 65). To our knowledge, only a single study has actually applied Rep-PCR to type Lactobacillus isolates from chickens (62) for the identification of potential probiotics to control Salmonella enterica serovar Enteritidis in egg-laying hens.The aim of this study was to investigate whether Rep-PCR could be used to analyze Lactobacillus species and strains in the chicken GIT. Several Rep-PCR techniques REP-, ERIC-, and (GTG)₅-PCR] were compared for the ability to type strains of several Lactobacillus species commonly isolated from the chicken GIT (L. crispatus, L. gallinarum, L. johnsonii, and L. reuteri). ERIC-PCR was able to simultaneously type isolates to the species and strain levels, and its strain differentiation ability was comparable with that of PFGE. ERIC-PCR was further applied to high-throughput analysis of a large number of isolates collected from chickens raised under NE and commercial conditions.