Detection and differentiation of chlamydiae by fluorescence in situ hybridization

Abstract

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and PARACHLAMYDIA: The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.

FIG. 1.

16S rRNA-based neighbor-joining tree showing the phylogenetic relations of representative members of the Chlamydiales. The specificities of oligonucleotide probes designed and tested for FISH are indicated. The taxonomy based on the reclassification of the Chlamydiales by Everett et al. (13) was used. Arrow, to outgroup; bar, 10% estimated evolutionary distance.

FIG. 2.

In situ identification of C. pneumoniae in HeLa 229 cells by FISH using Cy5-labeled probe Cpn-974, specific for C. pneumoniae (A) (showing an infra-red fluorescence, assigned to blue color); FLUOS-labeled probe Chlae-574, which targets all members of the family Chlamydiaceae (B) (green); and Cy3-labeled probe Chls-523, which hybridizes to all chlamydiae and chlamydia-like bacteria (C) (red). (D) Due to the overlap of colors, the chlamydial inclusions, which bound all three probes, appear white in the composite image. Bar, 10 μm.

FIG. 3.

In situ identification of the chlamydia-like bacteria UWE25 within free-living amoebae by FISH using Cy3-labeled probe Bn9₆₅₈ specific for a subgroup of the Parachlamydiaceae (A) (red) and Cy5-labeled probe Chls-523, which targets all chlamydiae (B) (blue). The FLUOS-labeled probe EUK516, which hybridizes to most eukaryotes (C) (green), was used to stain the amoebal host cells. (D) The chlamydiae appear purple in the overlap image. Bar, 10 μm.

FIG. 4.

Coinfection of HeLa 229 cells with C. trachomatis and C. pneumoniae. The simultaneous application of FLUOS-labeled probe Ct-623 specific for C. trachomatis (A) (green) and Cy5-labeled probe Cpn-214 specific for C. pneumoniae (B) (blue) allows the differentiation between the two chlamydia species. The simultaneous use of the Cy3-labeled eukaryotic probe EUK516 (C) (red) clearly demonstrated that a single HeLa 229 cell can be infected with both chlamydiae (D). Bar, 10 μm.

FIG. 5.

Combination of FISH and DFA. The performance of DFA subsequent to FISH allows the identification of chlamydiae (by the rRNA-targeted oligonucleotide probes) simultaneously with the detection of chlamydial antigen (by antichlamydia antibodies). (A) Fluorescent signal derived from DFA using fluorescein-labeled antichlamydia-LPS antibodies. (B) Fluorescent signal derived from FISH with C. pneumoniae-specific probe Cpn-214 labeled with Cy3. (C) Fluorescent signal derived from DAPI staining of chlamydiae and nuclei of the HeLa 229 host cells. (D) Composite image. Bar, 10 μm.

FIG. 6.

Visualization of the developmental cycle of C. pneumoniae by FISH (using Cy3-labeled probe Cpn-214; FISH column) and comparison with electron microscopy (EM column) and DFA staining (using fluorescein-labeled antichlamydia antibodies; DFA column). Three different points of time postinfection are shown. Arrows, chlamydial inclusions. Bars, 10 μm.