Growth and Development of Tetracycline-Resistant Chlamydia suis †

Tetracycline (TET) is a front-line antibiotic for the treatment of chlamydial infections in both humans and animals, and the emergence of TET-resistant (Tet r ) Chlamydia is of significant clinical importance. Recently, several Tet r chlamydial strains have been isolated from swine ( Sus scrofa) raised in production facilities in Nebraska. Here, the intracellular development of two Tet r strains, R19 and R27, is characterized through the use of tissue culture and immunofluorescence. The strains grow in concentrations of up to 4 m g of TET/ml, while a TET-sensitive (Tet s ) swine strain (S45) and a strain of the human serovar L2 (LGV-434) grow in up to 0.1 m g of TET/ml. Although inclusions form in the presence of TET, many contain large aberrant reticulate bodies (RBs) that do not differentiate into infectious elementary bodies. The percentage of inclusions contain- ing typical developmental forms decreases with increasing TET concentrations, and at 3 m g of TET/ml 100% of inclusions contain aberrant RBs. However, upon removal of TET the aberrant RBs revert to typical RBs, and a productive developmental cycle ensues. In addition, inclusions were found that contained both C. suis R19 were inverted onto a drop of Vectashield mounting medium (Vector Laboratories, Burlingame, Calif.) on a microscope slide. Fluorescent and differential interfer- ence contrast (DIC) images were collected and examined on a Leica DMLB microscope equipped with appropriate fluorescence filters. Images were col- lected digitally using a Spot Camera (Diagnostic Instruments, Sterling Heights, Mich.) and processed with Adobe Photoshop 5.0 (Adobe Software; San Jose, Calif.). Production of peptide antibody against R19 MOMP. Rabbit antiserum against a peptide (CGAGKVEDKGSAGELC) in the strain R19 major outer membrane protein (MOMP) was produced by Genemed Synthesis, Inc. (San Francisco, Calif.). The peptide was linked to keyhole limpet hemocyanin and administered in complete Freund’s adjuvant. Three subsequent booster injections were given with incomplete Freund’s adjuvant, followed by enzyme-linked immunosorbent assay analysis to confirm the specificity and relative concentration of the anti- body.