Induction of the Chlamydia muridarum Stress/Persistence Response Increases Azithromycin Treatment Failure in a Murine Model of Infection

Abstract

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...

Chlamydial Pre-Infection Protects from Subsequent Herpes Simplex Virus-2 Challenge in a Murine Vaginal Super-Infection Model

“…By day 9 pci, chlamydial shedding, while still detectable, has declined significantly from peak shedding at day 3 or day 6 pci. By day 27 pci, chlamydial shedding is no longer detectable [ 37 ]. In mice super-infected with HSV-2 on day 9 pci, protection was still observed and the super-infected mice (Cm-9D-H) exhibited significantly higher survival compared to the HSV-2 singly-infected mice (p<0.005), although survival in the Cm-9D-H group dropped slightly to 90% ( Fig 3B ).…”

Chronic Chlamydial Diseases: From Atherosclerosis to Urogenital Infections

“…If the chlamydial persistent state is, in fact, present in vivo, both detection by culture (persistent chlamydiae are by definition non-cultivable) and treatment of chlamydial infection may be impacted. Persistent chlamydiae are resistant to killing by antibiotics in vitro; and a model of amoxicillin induced C. muridarum persistence in mice indicates persistent chlamydiae are resistant to killing by azithromycin in vivo as well [99]. Furthermore, clinically relevant concentrations of commonly used penicillins induce C. trachomatis persistence in vitro [100].…”

Treatment challenges for urogenital and anorectal Chlamydia trachomatis

“…The meta-analyses of urogenital treatment efficacy found that the efficacy for azithromycin was lower for those with symptomatic infection compared with doxycycline [ 18 ]. It is unclear why this is so and suggests that perhaps a longer duration of azithromycin may be needed [ 34 ] with animal studies suggesting chlamydia shedding was higher in those which were persistently infected and that extended courses can overcome persistent infections [ 35 ].…”

“…A recent in vitro study examining the impact of β-lactam antibiotics on chlamydia persistence [ 39 ] found that all penicillins tested induced the formation of ABs with a 95 % reduction in chlamydia’s infectivity. Upon removal of the antibiotics, the chlamydia became infectious again, but β-lactam-induced persistent chlamydia was less susceptible to azithromycin in vitro [ 35 ]. Therefore, the question begs whether the marked increase in the use of beta-lactam antibiotics in recent years, [ 42 ] including its use in treating increases numbers of syphilis infections among gay men, [ 43 ] is contributing to antibiotic-induced persistence and whether increasing the duration of treatment can overcome this persistence [ 34 ] as has been demonstrated in animals [ 35 ].…”