Design and evaluation of 16S rRNA-targeted peptide nucleic acid probes for whole-cell detection of members of the genus Listeria

Abstract

Six fluorescein-labeled peptide nucleic acid oligomers targeting Listeria-specific sequences on the 16S ribosomal subunit were evaluated for their abilities to hybridize to whole cells by fluorescence in situ hybridization (FISH). Four of these probes yielded weak or no fluorescent signals after hybridization and were not investigated further. The remaining two FISH-compatible probes, LisUn-3 and LisUn-11, were evaluated for their reactivities against 22 Listeria strains and 17 other bacterial strains belonging to 10 closely related genera. Hybridization with BacUni-1, a domain-specific eubacterial probe, was used as a positive control for target accessibility in both Listeria spp. and nontarget cells. RNase T1 treatment of select cell types was used to confirm that positive fluorescence responses were rRNA dependent and to examine the extent of nonspecific staining of nontarget cells. Both LisUn-3 and LisUn-11 yielded rapid, bright, and genus-specific hybridizations at probe concentrations of approximately 100 pmol ml(-1). LisUn-11 was the brightest probe and stained all six Listeria species. LisUn-3 hybridized with all Listeria spp. except for L. grayi, for which it had two mismatched bases. A simple ethanolic fixation yielded superior results with Listeria spp. compared to fixation in 10% buffered formalin and was applicable to all cell types studied. This study highlights the advantages of peptide nucleic acid probes for FISH-based detection of gram-positive bacteria and provides new tools for the rapid detection of Listeria spp. These probes may be useful for the routine monitoring of food production environments in support of efforts to control L. monocytogenes.

FIG. 1.

Typical PNA hybridization results. (A) Cells of Bacillus cereus ATCC 11778 hybridized with the universal bacterial probe BacUni-1. (B) Cells of Listeria monocytogenes FSL-C1-122 hybridized with the Listeria-specific probe LisUn-11. Cells were fixed in a 50:50 mixture of absolute ethanol and PBS. Hybridizations were carried out as described in Materials and Methods.

FIG. 2.

Sequence variation in the 16S rRNA genes of Listeria and related genera. Alignments of partial sequences corresponding to the regions targeted by LisUn-3 (a) and LisUn-11 (b) are shown. The sequence of each probe is provided above the corresponding alignment. Residues differing from those found in the sequence of L. monocytogenes are boxed. For LisUn-3, the following GenBank accession numbers were aligned: X56153 (L. monocytogenes), X56151 (L. ivanovii), X56152 (L. innocua), X56148 (L. seeligeri), X56149 (L. welshimeri), X56150 (L. grayi), M58798 (Brochothrix thermosphacta), X60646 (Bacillus subtilis), X68417 (S. aureus), L14326 (G. haemolysans), X70321 (Kurthia zopfii), AJ301831 (E. faecalis), Z73313 (Carnobacterium sp.), and AF302116 (L. fermentum). The same sequences were aligned for LisUn-11, with the following exceptions: U84150 (L. monocytogenes), X98531 (L. seeligeri), and X56155 (B. thermosphacta). These alternate sequences were used to avoid sequence errors in X56153, X56148, and M58798 occurring in the region targeted by this probe. Abbreviations: Cbx, carboxyl terminus of probe, analogous to the 3′ terminus of DNA; Am, amino terminus of probe, analogous to the 5′ terminus of DNA; O, 8-amino-3,6-dioxaoctanoic acid linker; Flu, fluorescein.

FIG. 3.

Flow cytometric comparison of cell fixation protocols. Cells from the same overnight culture of L. monocytogenes strain Scott A were fixed using either formalin or ethanol, as described in the text. In these histograms, cell number is plotted against probe-conferred fluorescence. The rightward shift and narrower distribution for the ethanol-fixed population (solid histogram) shows that this method resulted in brighter and more homogeneous hybridizations than did formalin-based fixation (dashed histogram).

FIG. 4.

Combination of PNA-FISH and flow cytometry for the detection of Listeria in mixed culture. This experiment demonstrates the ability of these probes to clearly differentiate Listeria spp. from nontarget flora in cell mixtures. A 1-ml sample was removed from a mixed culture of L. monocytogenes Scott A and Lactobacillus fermentum growing at 30°C in a nonselective medium (MRS broth). The sample was pelleted and then fixed for 15 min in a 50:50 mixture of ethanol and PBS. A 100-μl portion of this sample was hybridized for 30 min with 200 pmol ml⁻¹ LisUn-11 and then analyzed by flow cytometry as described in Materials and Methods. Combined PNA-FISH and flow cytometry allowed a small number of L. monocytogenes cells (16% of total events collected) to be detected against a large background of L. fermentum (84% of total events collected).