Limits...
Discovering and differentiating new and emerging clonal populations of Chlamydia trachomatis with a novel shotgun cell culture harvest assay.

Somboonna N, Mead S, Liu J, Dean D - Emerging Infect. Dis. (2008)

Bottom Line: We developed a novel shotgun cell culture harvest assay for this purpose because we found that recent clinical samples do not form plaques.Clones were strain-typed by using outer membrane protein A and 16S rRNA sequences.Three clonal variants of reference strain Ba/Apache-2 were also identified.

View Article: PubMed Central - PubMed

Affiliation: Children's Hospital, Oakland Research Institute, Oakland, California, USA.

ABSTRACT
Chlamydia trachomatis is the leading cause of preventable blindness and bacterial sexually transmitted diseases worldwide. Plaque assays have been used to clonally segregate laboratory-adapted C. trachomatis strains from mixed infections, but no assays have been reported to segregate clones from recent clinical samples. We developed a novel shotgun cell culture harvest assay for this purpose because we found that recent clinical samples do not form plaques. Clones were strain-typed by using outer membrane protein A and 16S rRNA sequences. Surprisingly, ocular trachoma reference strain A/SA-1 contained clones of Chlamydophila abortus. C. abortus primarily infects ruminants and pigs and has never been identified in populations where trachoma is endemic. Three clonal variants of reference strain Ba/Apache-2 were also identified. Our findings reflect the importance of clonal isolation in identifying constituents of mixed infections containing new or emerging strains and of viable clones for research to more fully understand the dynamics of in vivo strain-mixing, evolution, and disease pathogenesis.

Show MeSH

Related in: MedlinePlus

Photographs and optical microscopy views of the wells showing plaques formed by Chlamydia trachomatis F/IC-Cal3 (A, C, E) and no plaque formed by clinical F persistent strain (B, D, F). A) Single well showing 2 distinct plaques (indicated by arrows). B) Well showing no plaque morphology. C) and D) Optical microscopy image showing plaque areas with little or no neutral red staining (arrows) surrounded by viable cells stained red (magnification ×100). Higher magnification (×400) showed numerous cells that had been infected by reference strain F/IC-Cal3 (E) and the clinical persistent F strain (F).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2570839&req=5

Figure 1: Photographs and optical microscopy views of the wells showing plaques formed by Chlamydia trachomatis F/IC-Cal3 (A, C, E) and no plaque formed by clinical F persistent strain (B, D, F). A) Single well showing 2 distinct plaques (indicated by arrows). B) Well showing no plaque morphology. C) and D) Optical microscopy image showing plaque areas with little or no neutral red staining (arrows) surrounded by viable cells stained red (magnification ×100). Higher magnification (×400) showed numerous cells that had been infected by reference strain F/IC-Cal3 (E) and the clinical persistent F strain (F).

Mentions: Plaques were visualized as a central area of cellular debris surrounded by viable infected cells with red staining of cytoplasm at the cell periphery (Figure 1, panels A and C). Inclusion bodies and nonviable cells remained clear. Any plaque (≈1–2 mm) that was clearly isolated from another plaque, or appeared as a solitary plaque in a well, was selected. A blunt-ended transfer pipette was used to punch a hole ≈1–2 mm in diameter through the gels over the plaque. The contents were placed into a microcentrifuge tube containing CMGH, sonicated and added to shell vials containing McCoy monolayers for propagation. Centrifugation of shell vials at 2,400 × g for 1 h at 35°C was required to successfully grow each clonally segregated strain. Strains were propagated and purified using gradient ultracentrifugation as previously described (2,18–20). The pellet was resuspended in SPG, and stored at –80°C.


Discovering and differentiating new and emerging clonal populations of Chlamydia trachomatis with a novel shotgun cell culture harvest assay.

Somboonna N, Mead S, Liu J, Dean D - Emerging Infect. Dis. (2008)

Photographs and optical microscopy views of the wells showing plaques formed by Chlamydia trachomatis F/IC-Cal3 (A, C, E) and no plaque formed by clinical F persistent strain (B, D, F). A) Single well showing 2 distinct plaques (indicated by arrows). B) Well showing no plaque morphology. C) and D) Optical microscopy image showing plaque areas with little or no neutral red staining (arrows) surrounded by viable cells stained red (magnification ×100). Higher magnification (×400) showed numerous cells that had been infected by reference strain F/IC-Cal3 (E) and the clinical persistent F strain (F).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2570839&req=5

Figure 1: Photographs and optical microscopy views of the wells showing plaques formed by Chlamydia trachomatis F/IC-Cal3 (A, C, E) and no plaque formed by clinical F persistent strain (B, D, F). A) Single well showing 2 distinct plaques (indicated by arrows). B) Well showing no plaque morphology. C) and D) Optical microscopy image showing plaque areas with little or no neutral red staining (arrows) surrounded by viable cells stained red (magnification ×100). Higher magnification (×400) showed numerous cells that had been infected by reference strain F/IC-Cal3 (E) and the clinical persistent F strain (F).
Mentions: Plaques were visualized as a central area of cellular debris surrounded by viable infected cells with red staining of cytoplasm at the cell periphery (Figure 1, panels A and C). Inclusion bodies and nonviable cells remained clear. Any plaque (≈1–2 mm) that was clearly isolated from another plaque, or appeared as a solitary plaque in a well, was selected. A blunt-ended transfer pipette was used to punch a hole ≈1–2 mm in diameter through the gels over the plaque. The contents were placed into a microcentrifuge tube containing CMGH, sonicated and added to shell vials containing McCoy monolayers for propagation. Centrifugation of shell vials at 2,400 × g for 1 h at 35°C was required to successfully grow each clonally segregated strain. Strains were propagated and purified using gradient ultracentrifugation as previously described (2,18–20). The pellet was resuspended in SPG, and stored at –80°C.

Bottom Line: We developed a novel shotgun cell culture harvest assay for this purpose because we found that recent clinical samples do not form plaques.Clones were strain-typed by using outer membrane protein A and 16S rRNA sequences.Three clonal variants of reference strain Ba/Apache-2 were also identified.

View Article: PubMed Central - PubMed

Affiliation: Children's Hospital, Oakland Research Institute, Oakland, California, USA.

ABSTRACT
Chlamydia trachomatis is the leading cause of preventable blindness and bacterial sexually transmitted diseases worldwide. Plaque assays have been used to clonally segregate laboratory-adapted C. trachomatis strains from mixed infections, but no assays have been reported to segregate clones from recent clinical samples. We developed a novel shotgun cell culture harvest assay for this purpose because we found that recent clinical samples do not form plaques. Clones were strain-typed by using outer membrane protein A and 16S rRNA sequences. Surprisingly, ocular trachoma reference strain A/SA-1 contained clones of Chlamydophila abortus. C. abortus primarily infects ruminants and pigs and has never been identified in populations where trachoma is endemic. Three clonal variants of reference strain Ba/Apache-2 were also identified. Our findings reflect the importance of clonal isolation in identifying constituents of mixed infections containing new or emerging strains and of viable clones for research to more fully understand the dynamics of in vivo strain-mixing, evolution, and disease pathogenesis.

Show MeSH
Related in: MedlinePlus