Development of solution phase hybridisation PCR-ELISA for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in Nurmi-type cultures

Abstract

Nurmi-type cultures (NTCs), derived from the fermentation of caecal contents of specifically pathogen-free (SPF) birds, have been used successfully to control salmonella colonisation in chicks. These cultures are undefined in nature and, consequently, it is difficult to obtain approval from regulatory agencies for their use as direct fed microbials (DFMs) for poultry. Progress towards the generation of effective defined probiotics requires further knowledge of the composition of these cultures. As such, species-specific, culture-independent quantification methodologies need to be developed to elucidate the concentration of specific bacterial constituents of NTCs. Quantification of specific bacterial species in such ill-defined complex cultures using conventional culturing methods is inaccurate due to low levels of sensitivity and reproducibility, in addition to slow turnaround times. Furthermore, these methods lack selectivity due to the nature of the accompanying microflora.

This study describes the development of a rapid, sensitive, reliable, reproducible, and species-specific culture-independent, solution phase hybridisation PCR-ELISA procedure for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in NTCs. In this technique, biotin-labelled primers were designed to amplify a species-specific fragment of a marker gene of known copy number, in both species. Resulting amplicons were hybridised with a dinitrophenol (DNP)-labelled oligonucleotide probe in solution and were subsequently captured on a streptavidin-coated microtitre plate. The degree of binding was determined by the addition of IgG (anti-DNP)–horseradish peroxidase conjugate, which was subsequently visualised using a chromogenic substrate, tetramethylbenzidine. This novel quantitative method was capable of detecting E. faecalis and P. pentosaceus at levels as low as 5 CFU per PCR reaction.

Introduction

Nurmi-type cultures (NTCs), derived from the fermentation of the mature caecal contents of specifically pathogen-free birds, have been successfully used to control salmonella colonisation in poultry (Bolder et al., 1992, Maciorowski et al., 1997, Stern et al., 2001). Caecal contents, from which these cultures are derived, are undefined (Salanitro et al., 1974, Zhu et al., 2002). As such, it is difficult to obtain approval from regulatory agencies to use these preparations as direct fed microbials (DFMs) for poultry. In an attempt to generate defined probiotics, several research groups have identified bacterial strains present in undefined gut microbiota and have used them singly or in mixtures to protect chicks against salmonella colonisation (Soerjadi et al., 1978, Weinack et al., 1985, Wierup et al., 1988, Stravic et al., 1991, Stravic, 1992). However, no defined product as effective as undefined gut microflora is available (Mead, 2000). A logical strategy is to generate defined probiotics, which are based on the relative concentrations in which constituent bacteria are present in effective NTCs. As a result, techniques are required to efficiently quantify specific bacterial species in these cultures.

Enterococcus faecalis is a key species in the development of certain fermented foods around the world (Hagrass et al., 1991, Franz et al., 1999, Elotmani et al., 2002), as well as playing important biological functions in the animal gut (Klein, 2003). The application of E. faecalis as a competitive exclusion agent and probiotic has been well reviewed in the literature (Pereira and Gibson, 2002, DeVuyst et al., 2003, Hufnagel et al., 2003). Specifically, E. faecalis has been documented to exhibit probiotic effects in the protection of chicks against Salmonalla typhimurium colonisation (Soerjadi et al., 1978) and also has the ability to protect epithelial cells against S. typhimurium invasion in vitro (Wagner et al., 2002). The potential of Pediococcus pentosaceus as a probiotic has also been well recognised (Mishra and Lambert, 1996, Gardiner et al., 2004, Lei and Jakobsen, 2004).

The quantification of specific bacterial species, especially those with certain physiological functions, in complex intestinal microbial populations has mainly been performed until now by culture-dependent techniques such as CFU determination on selective media (Tannock, 1999, Niamsup et al., 2003). Limitations associated with conventional culturing methods include low sensitivities (Dutta et al., 2001), inability to detect unculturable bacteria and unknown species, slow turnabout time, and poor reproducibility (Huijsdens et al., 2002). In addition, as large differences have been noted in the growth rates and growth requirements of different species, quantification by culture is highly likely to be inaccurate (Huijsdens et al., 2002). Furthermore, because of the ill-defined nature of the accompanying microbiota in NTCs, enumeration of specific species in such populations often proves problematic (O'Sullivan, 2000).

To overcome the problems associated with culture-based methodologies, culture-independent PCR has been applied to the quantification of specific genes as markers for bacterial presence (Bach et al., 2002). In a recent study, products generated from the amplification of a specific marker gene from bacterial cells could be visualised using ethidium bromide staining at levels of 50 CFU per PCR reaction (Daly et al., 2002). However, an increased level of detection was achieved by hybridisation of the PCR product with a species-specific labelled probe. Microwell ELISA-like hybridisation methods have therefore been developed (Nagata et al., 1985, van der Vliet et al., 1993, Gutierrez et al., 1998) and form the basis of convenient detection and quantification systems. In a solid phase PCR-ELISA methodology, Escherichia coli was detected in milk at levels as low as 5 CFU per PCR reaction (Daly et al., 2002). This technique involved the capture of 5′ biotin-labelled amplicons on streptavidin-coated microtitre plates followed by the subsequent addition of a dinitrophenol (DNP)-labelled oligonucleotide probe to the plate where solid phase hybridisation occurred to detect the bound amplicons.

This study describes the development of a novel solution phase hybridisation PCR-ELISA system and its application in the detection and quantification of E. faecalis and P. pentosaceus species in two NTCs. The modification of PCR-ELISA to a solution phase hybridization method removed the necessity for separate amplicon attachment and DNP-labeled oligonucleotide probe binding steps as required in the solid phase approach, thus decreasing the turnabout time of the assay. A crucial requirement for this methodology to be analytically accurate was the confirmation of the species specificity and copy number of the targeted marker genes in both species.