In culture, exposure to penicillin and other stressors induce chlamydiae to enter a non-infectious but viable state termed persistence. Chlamydiae may reenter their normal developmental cycle after stressor removal. Though aberrant RB similar to those present in culture models of persistence have been observed within infected tissues, the existence of persistent chlamydiae has not been definitively demonstrated in vivo. As a result, the role of persistent organisms in pathogenesis is undefined. In order to establish an experimentally tractable model of in vivo persistence, C. muridarum vaginally-infected mice were gavaged with either water or amoxicillin (amox). Vaginal swabs were collected for chlamydial titration and RNA isolated for quantification of pre-16s rRNA. Uterine tissue was analyzed by transmission electron microscopy (TEM). Although amox-treatment reduced vaginal shedding by >99%, C. muridarum pre-16s rRNA accumulation was unchanged by treatment. These data indicate that the amox-exposed organisms were viable but not infectious. Furthermore, TEM analyses demonstrated that inclusions in amox-treated animals contained primarily large, aberrant RB, but those observed in untreated control animals were normal. Collectively, these data suggest that amoxicillin treatment induces C. muridarum to enter the persistent state in vivo. This model also represents the first experimentally tractable animal model of chlamydial persistence.

Viable but noninfectious (stressed/persistent) chlamydiae are more resistant to azithromycin (AZM) in culture than are organisms in the normal developmental cycle. Chlamydia muridarum-infected mice were exposed to amoxicillin to induce the organisms to enter the persistent/stressed state and subsequently treated with AZM. AZM treatment failure was observed in 22% of persistently infected mice, with an average of 321,667 inclusion-forming units (IFU) shed after AZM treatment. Productively infected mice had a 9% rate of AZM treatment failure and shed an average of 12,083 IFU. These data suggest that stressed chlamydiae are more resistant to frontline antichlamydial drugs in vivo. Chlamydial species exhibit a unique biphasic developmental cycle, interchanging between the infectious elementary body (EB) and the replicative, noninfectious reticulate body (RB). Internalized EBs form vacuoles that fuse to form an inclusion, the membrane-bound structure in which EBs transform to RBs. After multiple rounds of division, RBs condense to form EBs, which are released to infect new host cells. In culture, exposure to environmental insults, including immunological stressors like gamma interferon (IFN-␥) and beta-lactam antibiotics such as amoxicillin (AMX), induces chlamydiae to reversibly detour from this normal developmental cycle (1, 2), entering a noninfectious, viable state alternately termed persistence or the chlamydial stress response. Interestingly, aberrant bodies (ABs) have been observed in vivo in the absence of a known stressor, suggesting that host environmental fluctuations induce at least a small subset of infecting organisms to enter persistence (3). Recently, we demonstrated this state in AMX-stressed Chlamydia muridarum-infected BALB/c mice. In our model, AMX treatment decreased vaginal shedding of infectious chlamydiae by Ͼ99% without affecting viability. Shedding of infectious EBs resumed within 1 week after treatment cessation (4). These data demonstrate that AMX can be used to reversibly induce chlamydial persistence in vivo.In culture, persistent chlamydiae are refractory to treatment with first-choice antibiotics. Persistent/stressed C. trachomatis strains within penicillin-exposed Hec1B cells are resistant to treatment with azithromycin (AZM) (5), and IFN-␥ exposure makes C. trachomatis more resistant to doxycycline killing (6). Additionally, doses of AZM or ofloxacin up to 4ϫ the MIC are ineffective against persistent C. pneumoniae infection in culture (7). Thus, many investigators have proposed that persistent chlamydiae may contribute to chronic disease by evading antibiotic treatment. While there is evidence that persistent infection occurs in vivo (8, 9), the absence of an experimentally tractable animal model of viable but noninfectious chlamydial infection has hampered direct testing of this prediction. This study will seek to determine if persistent organisms are resistant to treatment by the first-choice antibiotic, AZM, in our newly characterized mouse model (4). All animal experiments descr...

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen, but more than 70% of patients fail to seek treatment due to the asymptomatic nature of these infections. Women suffer from numerous complications from chronic chlamydial infections, which include pelvic inflammatory disease and infertility. We previously demonstrated in culture that host cell nectin-1 knockdown significantly reduced chlamydial titers and inclusion size. Here, we sought to determine whether nectin-1 was required for chlamydial development in vivo by intravaginally infecting nectin-1-/- mice with Chlamydia muridarum and monitoring chlamydial shedding by chlamydial titer assay. We observed a significant reduction in chlamydial shedding in female nectin-1-/- mice compared to nectin-1+/+ control mice, an observation that was confirmed by PCR. Immunohistochemical staining in mouse cervical tissue confirmed that there are fewer chlamydial inclusions in Chlamydia-infected nectin-1-/- mice. Notably, anorectal chlamydial infections are becoming a substantial health burden, though little is known regarding the pathogenesis of these infections. We therefore established a novel male murine model of rectal chlamydial infection, which we used to determine whether nectin-1 is required for anorectal chlamydial infection in male mice. In contrast to the data from vaginal infection, no difference in rectal chlamydial shedding was observed when male nectin-1+/+ and nectin-1-/- mice were compared. Through the use of these two models, we have demonstrated that nectin-1 promotes chlamydial infection in the female genital tract but does not appear to contribute to rectal infection in male mice. These models could be used to further characterize tissue and sex related differences in chlamydial infection.