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. 2014 Sep;58(9):5413–5420. doi: 10.1128/AAC.03187-14

Maternal Intravenous Treatment with either Azithromycin or Solithromycin Clears Ureaplasma parvum from the Amniotic Fluid in an Ovine Model of Intrauterine Infection

Yuichiro Miura a, Matthew S Payne a, Jeffrey A Keelan a, Andres Noe a, Sean Carter a, Rory Watts a, Owen B Spiller b, Alan H Jobe c, Suhas G Kallapur c, Masatoshi Saito d, Sarah J Stock e, John P Newnham a, Matthew W Kemp a,✉
PMCID: PMC4135857  PMID: 24982089

Abstract

Intrauterine infection with Ureaplasma spp. is strongly associated with preterm birth and adverse neonatal outcomes. We assessed whether combined intraamniotic (IA) and maternal intravenous (IV) treatment with one of two candidate antibiotics, azithromycin (AZ) or solithromycin (SOLI), would eradicate intrauterine Ureaplasma parvum infection in a sheep model of pregnancy. Sheep with singleton pregnancies received an IA injection of U. parvum serovar 3 at 85 days of gestational age (GA). At 120 days of GA, animals (n = 5 to 8/group) received one of the following treatments: (i) maternal IV SOLI with a single IA injection of vehicle (IV SOLI only); (ii) maternal IV SOLI with a single IA injection of SOLI (IV+IA SOLI); (iii) maternal IV AZ and a single IA injection of vehicle (IV AZ only); (iv) maternal IV AZ and a single IA injection of AZ (IV+IA AZ); or (v) maternal IV and single IA injection of vehicle (control). Lambs were surgically delivered at 125 days of GA. Treatment efficacies were assessed by U. parvum culture, quantitative PCR, enzyme-linked immunosorbent assay, and histopathology. Amniotic fluid (AF) from all control animals contained culturable U. parvum. AF, lung, and chorioamnion from all AZ- or SOLI-treated animals (IV only or IV plus IA) were negative for culturable U. parvum. Relative to the results for the control, the levels of expression of interleukin 1β (IL-1β), IL-6, IL-8, and monocyte chemoattractant protein 2 (MCP-2) in fetal skin were significantly decreased in the IV SOLI-only group, the MCP-1 protein concentration in the amniotic fluid was significantly increased in the IV+IA SOLI group, and there was no significant difference in the histological inflammation scoring of lung or chorioamnion among the five groups. In the present study, treatment with either AZ or SOLI (IV only or IV+IA) effectively eradicated macrolide-sensitive U. parvum from the AF. There was no discernible difference in antibiotic therapy efficacy between IV-only and IV+IA treatment regimens relative to the results for the control.

INTRODUCTION

The mean global rate of preterm birth (PTB, defined as live delivery before 37 weeks of completed gestation) in 2010 was 11.1% (1), with an estimated 30 million babies born preterm annually. The PTB rate in most developed countries is around 8%, while in other parts of the world, the rate is 2- to 3-fold higher. In many countries, this rate has increased over the past 20 years (1). PTB is a leading cause of neonatal morbidity (2) and constitutes a significant financial burden on the health care system (3). Twenty-five to 30% of PTB is preceded by preterm prelabor rupture of membranes (PPROM) (2). Ureaplasma spp. are among the microorganisms most frequently isolated from the amniotic fluid (AF) and placental membranes following preterm labor and PPROM (4). The isolation of Ureaplasma spp. from the placenta or the lower genital tract in pregnancy is associated with both intrauterine inflammation and PTB (5, 6). Thus, Ureaplasma spp. are believed to play a causal role in the process of infection-driven PTB and a host of related neonatal morbidities (7). Accordingly, there is an urgent need to develop pharmaceutical regimens for the prevention and treatment of intrauterine Ureaplasma spp. infection in pregnancy.

Although maternal erythromycin (EM) remains a standard antibiotic treatment for PPROM, due to its efficacy and safety based on the findings of previous studies (8–10), there are many reports that suggest variable EM treatment efficacy and even a risk of adverse postnatal outcomes (11–13). One reason for the variable efficacy of EM treatment in PPROM is the poor transplacental transfer of EM after maternal oral administration. To eradicate intrauterine infection, it is vital to achieve therapeutic concentrations of antibiotics at the site of infection/colonization; thus, it is possible that direct intraamniotic (IA) administration of antibiotics may be more effective than intravenous (IV) dosing. However, given the small potential risk to fetal wellbeing posed by amniocentesis (14), as well as the cost of the procedure, IA dosing would only be justified by significant improvements in treatment efficacy. We previously demonstrated that IA administration of EM delivers a long-lasting, therapeutic dose of EM to the AF (15). However, IA administration of EM failed to completely resolve intrauterine infection with EM-sensitive Ureaplasma parvum in a sheep model of pregnancy, suggesting that more potent chemotherapeutic agents are required (16).

Azithromycin (AZ) is an expanded-spectrum macrolide antibiotic that has a similar structure to EM (17) but demonstrates better tissue penetration (17). AZ has low bioavailability (approximately 37%) (18), a prolonged half-life, and achieves high and sustained antibiotic tissue levels in pregnant women at term (19). Although cases of resistance have been reported and are potentially increasing (20, 21), AZ has been shown to be effective for eradicating intrauterine U. parvum infection in nonhuman primates (22), with a MIC of 0.5 to 4 μg/ml (23). AZ has been used extensively in pregnancy to treat a host of infections, including chlamydia (24), scrub typhus (25), and malaria (as part of combination therapy) (26). AZ is a readily available generic drug with an excellent safety profile; cases of hepatotoxicity are quite rare (27), and the small risk of Q-T interval prolongation identified in high-risk patients (28) is not significant in young or middle-aged individuals (29), the demographics into which most pregnant women fall. Trials of AZ in pregnancy to prevent preterm birth have, like those of many other antibiotics, been equivocal (30), and most studies to date suggest that transplacental transfer of AZ is poor. Pharmacokinetic studies in human pregnancy have reported that a single oral dose of AZ achieves peak AF concentrations of 151 ng/ml and fetal blood concentrations of 27 ng/ml, both well below the MIC range of 500 ng/ml for Ureaplasma spp. (19). Using a sheep model of pregnancy, we have shown that a single maternal IV administration of AZ fails to achieve levels above the MIC in the AF, although IA administration of AZ achieves levels above the MIC in the AF for 24 h or more (15). Interestingly, both of these studies demonstrated slow clearance of AZ from the AF, with a half-life of 17 to 30 h, suggesting that repeated dosing may achieve effective concentrations in the AF. Recent studies in nonhuman primates confirm this, showing that repeated IV doses of maternal AZ (25 mg/kg of body weight/day for 10 days) can result in effective concentrations of AZ in the AF, with clearance of AZ-sensitive U. parvum serovar 1 infection (95% effective concentration, 39 ng/ml) from the AF at 7 days (31).

Solithromycin (SOLI) is a new macrolide (fluoroketolide) antibiotic currently being investigated for the treatment of infections, including community-acquired bacterial pneumonia and urogenital gonorrhoeae (32). SOLI is acid stable, well tolerated, exhibits excellent oral bioavailability (∼70%), and demonstrates superior tissue accumulation (33). Importantly, all strains of U. parvum, Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma genitalium tested are sensitive to SOLI (MICs of <16 ng/ml) (34). SOLI is (i) the first of its class that has comparable efficacy and favorable safety relative to these parameters for levofloxacin (35), (ii) highly potent and active against many macrolide-resistant strains of susceptible organisms (36), and (iii) the first of its class to exhibit efficient maternal-to-fetal transfer, such that a single maternal dose provides maternal, amniotic, and fetal antimicrobial coverage for 24 h (37). SOLI has also been shown to exhibit significant anti-inflammatory effects in vitro and in vivo (38). SOLI thus appears to be a promising agent for use in the treatment of intrauterine infection. However, it has not yet been administered in pregnancy and the necessary safety studies in pregnant women have yet to be undertaken.

In the present study, we compared the ability of a 4-day course of either AZ or SOLI, delivered either maternally via IV injection or via IA injection, to eradicate intrauterine U. parvum serovar 3 infection in a sheep model of pregnancy. We hypothesized that the superior pharmacokinetics and greater potency of SOLI would result in eradication of IA U. parvum infection following maternal IV administration alone, whereas AZ would require combined IV and IA administration to achieve the same effects. We also hypothesized that eradication of infection would be accompanied by reduced expression levels of inflammatory cytokines and chemokines in fetal and amniotic tissues.

MATERIALS AND METHODS

Animals and tissue collection.

All procedures involving animals were performed in Western Australia following review and approval by the animal care and use committee of the University of Western Australia. Date-mated Australian merino ewes (term is 148 ± 2 days) were bred to carry single pregnancies. At the conclusion of each experimental protocol, the ewes were euthanized with intravenous pentobarbitone (100 mg/kg). The fetuses were then surgically delivered and euthanized with pentobarbitone (100 mg/kg). Tissues for U. parvum quantification and inflammatory analyses were snap-frozen. Fetal lungs (right upper lobes) were perfusion fixed in 10% neutral buffered formalin for 24 h before paraffin embedding. The chorioamnions were immersed in 10% neutral buffered formalin for 24 h before paraffin embedding. Blood gas and electrolyte analyses were performed on a Siemens Rapidlab 1200 platform (Siemens, Munich, Germany). Complete blood counts were performed by VetPath (Perth, WA, Australia).

IA administration of U. parvum and treatment.

IA inoculation with U. parvum serovar 3 was performed at 85 days of gestational age (GA) under ultrasound guidance, with successful IA targeting verified by electrolyte analysis (Cl−) of AF. The MICs for AZ and SOLI against this isolate were 1.0 μg/ml and 0.03 μg/ml, respectively. At 120 days of GA, animals were randomly assigned to receive one of the following treatments (all maternal IV drug regimens were 10 mg/kg of maternal weight, every 24 h for 4 days): (i) maternal IV SOLI with a single IA injection of vehicle given with the first IV injection (IV-only SOLI), (ii) maternal IV SOLI with a single IA SOLI injection (0.14 mg/kg of fetal weight) (IV+IA SOLI), (iii) maternal IV AZ and a single IA injection of vehicle (IV-only AZ), (iv) maternal IV AZ and a single IA AZ injection (1.4 mg/kg of fetal weight) (IV+IA AZ), or (v) maternal IV and single IA injections of vehicle (control). The dosages of AZ and SOLI used were derived from our previous pharmacokinetic studies in pregnant sheep (15, 37). In a previous study, 3 of 16 chronically catheterized fetuses administered IA SOLI at 1.4 mg/kg of estimated fetal weight died, with autopsy revealing bloody ascites and hepatomegaly. We were unable to confirm the cause of death (which may have been a function of the invasive nature of the protocol itself) and there was no evidence of toxicity in the other animals in this protocol, including eight additional animals receiving maternal IV SOLI only. Based on the high amniotic fluid and fetal plasma concentrations achieved in our previous work and SOLI's low MIC against U. parvum, the dose of IA SOLI administered in the present study was set at 1/10 that of IA AZ to limit any potential adverse effects on the fetus. Fetal weights were estimated according to their gestational ages. Saline was used as a vehicle for AZ treatments. A buffer comprised of 38 mM tartaric acid, 164 mM d-mannitol, 0.5% thioglycerol at pH 4.2 was used as the vehicle for all SOLI treatments. All fetuses were surgically delivered at 125 days of GA under terminal anesthetic. The treatment efficacies were assessed by the methodology outlined below.

Ureaplasma culture and infection screening.

U. parvum serovar 3 was cultured using 10B broth (Melbourne University Media Preparation Unit, Melbourne, Australia) and quantified by assessing U. parvum color change units (CCU) in a broth dilution series as previously described (39). Isolates in 10B broth were aliquoted across one row (eight wells, 450 μl in each) of a 2-ml deep-well microtiter plate. Fifty microliters of sample was added to the first well in each row, and 10-fold dilutions were performed across the plate, with the final well representing a 10−8 dilution of the original sample. The plates were sealed and incubated at 37°C, 5% CO2, 2% O2 for 48 h. Positive growth was detected by an alkaline pH shift (detected as a red color change) in the 10B broth, and samples were quantitated based upon the highest dilution in the row.

Nucleic acid extraction.

DNA was extracted from 250-μl samples of AF and cord blood plasma using a Siemens sample preparation kit 1.0 (Siemens) on a Kingfisher Duo extraction platform (Thermo Fisher Scientific, Inc., Waltham, MA, USA), following the manufacturer's instructions. All extracts were eluted in a final volume of 100 μl of elution buffer (Siemens).

Isolation of RNA from fetal tissues.

Total RNA was extracted from liquid nitrogen-homogenized fetal tissues (lung right lower lobe, axilla skin, chorioamnion, and spleen) using TRIzol (Life Technologies, Carlsbad, CA, USA) as previously described (40, 41). Extracted RNA was treated with Turbo DNase (Life Technologies) in accordance with the manufacturer's instructions to remove any residual DNA. The RNA template was quantified with a Qubit 2.0 fluorometer (Life Technologies), using a broad-range RNA quantitation kit (Life Technologies). RNA extracts were diluted in nuclease-free water (Life Technologies) to a final concentration of 25 ng/μl.

U. parvum nucleic acid detection.

U. parvum DNA in AF and cord blood plasma was detected using a real-time PCR assay targeting the urease gene of U. parvum as described by Yi et al. (42) on a ViiA 7 real-time PCR system (Life Technologies). The reaction mixtures contained (final volume, 20 μl) 1× TaqMan fast advanced master mix, 0.9 μM primers UU1613F and UU1524R, 0.25 μM probe UU-parvo (6-carboxyfluorescein [FAM]), 5 μl template DNA, and nuclease-free water (all from Life Technologies). The reaction cycling conditions were as follows: initial denaturation/Taq activation at 95°C for 20 s, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s (data acquisition phase).

qPCR. (i) U. parvum RNA.

The quantitative PCR (qPCR) reaction mixtures for U. parvum RNA contained (final volume, 20 μl) 10 μl Express SuperScript qPCR supermix universal, 2 μl Express SuperScript mix for one-step qPCR, 50 nM ROX reference dye, 0.9 μM primers UU1613F and UU1524R, 0.25 μM probe UU-parvo (FAM), 125 ng total RNA template, and nuclease-free water (all from Life Technologies). The reaction cycling conditions were as follows: 15 min of reverse transcription at 50°C and an initial denaturation/polymerase activation at 95°C for 20 s, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s (data acquisition phase). The U. parvum RNA levels were quantitated with ViiA7 real-time PCR system 1.2.1 software (Life Technologies), using a standard curve (R2 > 0.99) derived from U. parvum control RNA at 0.2 ng, 0.02 ng, 0.002 ng, and 0.0002 ng per 20-μl reaction mixture.

(ii) Cytokines and chemokines.

Ovine-specific PCR primers and hydrolysis probes for interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein 2 (MCP-2) (all Life Technologies) were used to perform quantitative PCR for cytokines and chemokines. The reactions were performed using an Express one-step SuperScript quantitative reverse transcription-PCR kit (Life Technologies) with 125 ng of template fetal tissue RNA in a total volume of 20 μl according to the manufacturer's instructions. The reaction cycling conditions were as described above for U. parvum RNA analysis. The target quantification cycle (Cq) values were normalized to the 18S rRNA Cq value and expressed as fold changes relative to the value for the pooled control. The reaction efficiencies were within the limits proposed in the MIQE guidelines (43). dCq values were used to perform statistical analyses for significant differences between values for intervention groups versus values for the control group.

Enzyme-linked immunosorbent assays (ELISAs).

Quantification of MCP-1 protein concentrations in AF samples was performed using a commercial kit (VS0081S-02) from Kingfisher Biotech (St. Paul, MN, USA). The samples were incubated overnight (16 h) at 4°C. The remainder of the assay was performed in accordance with the manufacturer's instructions.

Fetal lung histology.

Five-micrometer paraffin-embedded sections of 10% (pH 7.4) formalin-fixed fetal lungs (right upper lobe) were stained with Meyer's hemotoxylin and eosin. Qualitative scoring of airspace infiltration was performed by a single investigator blinded to treatment groups. Six fields (×200 total magnification) were scored for each animal. Airspace infiltration and consolidation were graded as follows: 0, no inflammatory cells in airspace; 1, airspace inflammatory cells but no consolidation; 2, airspace inflammatory cells and limited microconsolidation (1 to 4 per field) foci; 3, airspace inflammatory cells and numerous (>4 per field) but predominantly discrete microconsolidation foci; and 4, airspace inflammatory cells and confluent airspace consolidation.

Chorioamnion histology.

Five-micrometer paraffin-embedded sections of 10% (pH 7.4) formalin-fixed chorioamnion were stained with Meyer's hemotoxylin and eosin. Qualitative scoring of histologic chorioamnionitis was performed by a single investigator blinded to treatment groups, using a modified Redline staging system as described previously (16, 44, 45). Briefly, the tissue plane and severity of inflammatory cell infiltration were graded as follows: 0, no inflammatory cells identifiable/no chorioamnionitis; 1, stage 1, grade 1 chorioamnionitis; 2, stage 1, grade 2 chorioamnionitis; 3, stage 2, grade 2 chorioamnionitis; and 4, stage 3, grade 2 chorioamnionitis.

Statistical analysis.

All values reported represent the group mean ± standard deviation (SD). Analyses were performed using IBM SPSS for Windows, version 20.0 (IBM Corporation, Armonk, NY, USA). Data were tested for normality using the Shapiro-Wilk test. For normally distributed data, mean differences were tested for significance using one-way analysis of variance (ANOVA) with a P value of <0.05 accepted as significant. Multiple post hoc comparisons were performed using Tukey's test. Between-group differences in nonparametric data were tested for significance with Kruskal-Wallis one-way ANOVA, with a P value of <0.05 accepted as significant. Multiple post hoc comparisons were performed using the rank sum test with a P value corrected for n multiple comparisons.

RESULTS

U. parvum culture and DNA and RNA analysis.

There was no significant difference between treatment and control groups for any physiological or hematological variable at fetal delivery (Table 1). AF from all AZ- or SOLI-treated animals (IV only or IV+IA) were negative for culturable U. parvum, while those from all control animals contained culturable U. parvum (2.8 × 105 ± 4.0 × 105 CCU U. parvum/ml [mean ± SD]). The lung and chorioamnion samples from all control animals contained culturable U. parvum (2.2 × 103 ± 4.4 × 103 and 4.1 × 103 ± 5.4 × 103 CCU U. parvum/ml, respectively), while those from all AZ- or SOLI-treated animals (IV only or IV+IA) were negative for culturable U. parvum (Table 1). All AF samples had detectable U. parvum DNA. Sixty percent (3/5) of animals from the saline control group, 14% (1/7) of those from the IV+IA AZ group, and none of the SOLI-treated animals were PCR positive for U. parvum DNA in cord blood plasma (data not shown). Significant (P < 0.01) reductions in skin U. parvum RNA levels were detected in all animals in antibiotic-treated groups compared to the levels in control animals (Fig. 1A). No significant reduction in chorioamnion (Fig. 1B) and lung U. parvum RNA levels (Fig. 1C) was detected in any animals in the four treatment groups relative to the levels in control animals. U. parvum RNA was detected in only two spleen samples, both in the control group (data not shown).

TABLE 1.

Fetal data at delivery and U. parvum culture quantitation in AF, lung, and chorioamniona

Group Delivery wt (kg) Quantity of U. parvum (CCU/ml) cultured from:
 Value for indicated blood test in CB
AF (×105) Lung (×103) Chorioamnion (×103) WBC (×103/μl) Neutrophils (×103/μl) Hb (g/liter)
Control (n = 5) 2.9 ± 0.2 2.8 ± 4.0 2.2 ± 4.4 4.1 ± 5.4 3.1 ± 1.5 0.7 ± 0.8 126.2 ± 12.5
IV SOLI (n = 8) 2.9 ± 0.4 0.0 ± 0.0‡ 0.0 ± 0.0‡ 0.0 ± 0.0‡ 2.9 ± 0.7 0.4 ± 0.2 131.5 ± 15.3
IV+IA SOLI (n = 8) 3.0 ± 0.4 0.0 ± 0.0‡ 0.0 ± 0.0‡ 0.0 ± 0.0‡ 3.7 ± 1.1 0.3 ± 0.1 129.0 ± 12.7
IV AZ (n = 7) 2.9 ± 0.4 0.0 ± 0.0‡ 0.0 ± 0.0‡ 0.0 ± 0.0‡ 4.8 ± 1.9 1.0 ± 0.9 130.8 ± 8.5
IV+IA AZ (n = 7) 3.0 ± 0.2 0.0 ± 0.0‡ 0.0 ± 0.0‡ 0.0 ± 0.0‡ 3.4 ± 1.8 0.7 ± 0.9 129.7 ± 12.8
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a

All values are means ± SD. CB, cord blood; WBC, white blood cells; Hb, hemoglobin. Significant differences versus value for saline-treated control animals are indicated: ‡, P < 0.01.

FIG 1.

FIG 1

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Quantitative PCR detection of U. parvum RNA in fetal skin (A), chorioamnion (B), and fetal lung (C). The diamonds show the U. parvum RNA values. The bars show median U. parvum RNA values in each group. Significant differences versus value for saline-treated control are indicated: ‡, P < 0.01.

Cytokine and chemokine mRNA expression.

There was no significant difference between animals in the treatment and control groups for fetal chorioamnion (data not shown) or lung cytokine/chemokine expression (Fig. 2A). Significant reductions in fetal skin IL-1β (0.4 ± 0.4 versus 1.1 ± 0.4, P < 0.05), IL-6 (0.4 ± 0.2 versus 1.0 ± 0.2, P < 0.05), IL-8 (0.3 ± 0.6 versus 1.2 ± 0.7, P < 0.01), and MCP-2 (0.3 ± 0.3 versus 1.8 ± 2.4, P < 0.05) mRNA expression levels were detected in the IV SOLI-only group compared to the levels in control animals. A significant reduction in skin IL-6 was also detected in the IV+IA SOLI group compared to the levels in control animals (0.5 ± 0.1 versus 1.0 ± 0.2, P < 0.01) (Fig. 2B). No significant reduction in AF MCP-1 protein concentration was detected in any animals in the treatment groups compared to the levels in animals in the control group, while a significant increase was detected in animals in the IV+IA SOLI group compared to the levels in control group animals (266.7 ± 93.6 versus 121.1 ± 52.4 pg/ml) (Fig. 3).

FIG 2.

FIG 2

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Relative expression of cytokine/chemokine mRNAs in fetal lung (A) and fetal skin (B), measured with quantitative PCR. All values are the group mean normalized expression versus the value for pooled saline-treated control. Significant differences versus value for saline-treated control are indicated: ‡, P < 0.01; *, P < 0.05.

FIG 3.

FIG 3

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MCP-1 concentrations in AF. Significant difference versus value for saline-treated control is indicated: *, P < 0.05.

Histology.

There were no significant differences in qualitative airspace inflammation scoring among the five groups, although animals from the IV SOLI group scored lowest overall (Fig. 4). With respect to the chorioamnion, all animals in the saline-treated control group exhibited significant chorioamnionitis (score of 4) and there were no significant differences in qualitative chorioamnion inflammation scoring among the five groups (Fig. 5).

FIG 4.

FIG 4

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Qualitative scoring of histological inflammation in fetal airspace. Images are representative of the indicated inflammatory scores (2 to 4) assigned to each field assessed (×200 total magnification). There was no sample scored as 1 in our experimental group. Scale bar = 100 μm. Mean value ± SD is shown for each treatment.

FIG 5.

FIG 5

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Qualitative scoring of histological inflammation of chorioamnion. Images are representative of the indicated scores (1 to 4) in the modified Redline scoring system used to score each field assessed (×200 total magnification). Scale bar = 100 μm. Mean value ± SD is shown for each treatment.

DISCUSSION

In this study, we made three important observations. First, treatment with either AZ or SOLI, regardless of the route of administration, effectively eradicated intrauterine U. parvum infection, eliminating culturable U. parvum from the AF, lung, and chorioamnion. The reduction in culturable U. parvum was accompanied by significant reductions in U. parvum RNA levels in skin samples. No significant difference in U. parvum RNA levels in chorioamnion and lung samples among the five groups was demonstrable. However, viewed from another perspective, lung U. parvum RNA was detectable by qPCR in only 63% (19/30) of treated animals in the present study (100% of control animals), whereas using identical detection methods, our previous work showed the presence of lung U. parvum RNA in all IA erythromycin (EM) treatment groups (16). Collectively, these results demonstrate the superior antimicrobial efficacy of AZ or SOLI compared to that of EM in this model. The AZ treatment efficacy data in the present study are also consistent with the findings of recent work in nonhuman primate models of intrauterine infection (22).

Second, after 4 days of treatment, there was no difference in the viable U. parvum loads in AF or fetal tissues from the IV-only or IV+IA treatment groups, suggesting that no additional benefit was gained from the IA administration of antibiotics at the 4-day time point. While this was somewhat anticipated for the SOLI treatment groups, due to its effective maternal-amniotic transfer, we did expect that the additional IA dose of AZ would result in enhanced bacterial elimination due to our previous findings of subtherapeutic levels of AZ in the AF and fetal circulation following a single maternal IV dose (15). Presumably the accumulation of AZ in the AF following repeated daily doses, as reported recently by Acosta et al. (31), is responsible for this observation. It should, however, be noted that in the present study, we did not assess the rate at which each treatment modality resolved intrauterine U. parvum infection. It is possible that the superior maternal-fetal transfer of SOLI (37) and its greater potency against U. parvum may mean that a SOLI-based treatment regimen will allow for more rapid resolution of intrauterine infection relative to the results with AZ treatment. As rapid resolution of intrauterine U. parvum infection is likely to limit inflammation-associated injury to the fetus and reduce the risk of inflammation-driven PTB, further studies to assess the U. parvum clearance rate achieved with each agent are warranted.

Third, we found that antibiotic treatment did not result in widespread reductions in amniotic or fetal tissue inflammation relative to the levels in saline-treated controls. Significant reductions in cytokine/chemokine mRNA levels relative to the levels in the saline-treated controls were only observed in the skin following IV-only SOLI treatment. Similar reductions were not detected in lung or chorioamnion samples (both key mediators of intrauterine inflammation), and there was no significant difference in lung and chorioamnion histological scoring across all five treatment groups. Conversely, animals in the IA+IV SOLI treatment group had significant increases in AF MCP-1 expression relative to the levels in animals receiving the saline control. These data were somewhat unexpected given recent data demonstrating SOLI's marked anti-inflammatory effects in in vitro and in vivo studies (38). As such, we conclude that the administration of antibiotics alone may not be sufficient to resolve intrauterine inflammation. Again these data are consistent with previous work undertaken in nonhuman primates, as well as data from clinical studies (11, 46, 47).

As intrauterine inflammation is hypothesized to be a primary mediator of infection-associated PTB and fetal injury (48), suppression of pathological intrauterine inflammation is likely to be required for effective prevention of PTB and adverse neonatal outcomes. Thus, additional anti-inflammatory therapy may be needed to prevent PTB. We have recently demonstrated the significant suppressive effect of IA administration of polymyxin B (49) and cytokine-suppressive anti-inflammatory drugs (50) against intrauterine inflammation. In addition, Gravett et al. reported significantly delayed age at birth and reduction in IA inflammatory cytokine/chemokine levels due to a combination of antibiotics, glucocorticoids, and nonsteroidal anti-inflammatory drugs (47). We suggest that experiments to explore the efficacy of combining efficient antibiotics with anti-inflammatory agents against intrauterine U. parvum infection are now warranted.

U. parvum DNA was detected in all AF samples, irrespective of treatment randomization. This result is consistent with the interpretation that U. parvum DNA persists after the microorganism has been cleared, as previously reported (22). As such, quantitation of U. parvum DNA does not appear to be a suitable method to assess the efficacy of antibiotic treatment; the use of an RNA-based approach is likely to be more informative. Additionally, the detection of U. parvum DNA in cord blood plasma and U. parvum RNA in the spleen suggests that U. parvum either has the capacity to invade the systemic fetal circulation or is specifically phagocytosed (perhaps as part of the fetal adaptive immune response). This observation is also consistent with previous studies demonstrating the likely importance of U. parvum to preterm birth and adverse neonatal outcomes (7, 16, 51–53).

Although the sheep has long proven an appropriate model for infection in human pregnancy, this study has several limitations that should be considered when interpreting these findings. The ovine placenta differs both structurally and metabolically from the human placenta (54). In the sheep, SOLI is metabolized to form two bioactive metabolites at a significantly higher rate than in the human; these metabolites accumulate in the AF and likely contribute to its extended antimicrobial activity (37). We did not analyze the concentrations of AZ and SOLI in AF, maternal blood, and fetal blood, so we were unable to determine the extent of transfer and accumulation of the parent drugs and their metabolites in the various compartments. Our previous studies have measured maternal-fetal biodistribution of AZ and SOLI in this model, but only over a 72-h period following a single dose (15, 37). The present study also employed IV antibiotic administration; achieving equivalent intraamniotic AZ concentrations using this route would likely require a significantly higher dose if given orally, which may not be well tolerated due to gastrointestinal effects. Due to a lack of appropriate samples, we were unable to analyze the effect of sustained macrolide exposure on the fetal liver, meaning that any potential toxicity of AZ or SOLI could not be adequately determined.

Despite these limitations, it should also be noted that our findings are consistent with earlier reports involving nonhuman primates and also with human studies, as described above. These data demonstrate that the administration of either AZ or SOLI is more effective than EM in treating intrauterine U. parvum infection in a sheep model of pregnancy and suggest that a 4-day maternal course of either antibiotic, at a dose sufficient to replicate the IV dosing employed here, is sufficient to eradicate culturable U. parvum from the AF.

The detection of U. parvum RNA in fetal lung tissue (suggestive of viable U. parvum) from 19 of 30 antibiotic-treated animals suggests the potential for recolonization of the AF had the animals been monitored past 125 days of GA. Complete clearance of infection is likely to be important to prevent PTB and associated neonatal morbidities while minimizing the risk of antibiotic resistance developing. As such, future studies should seek to identify the minimum length of antibiotic treatment required to result in continued resolution of infection well past the cessation of antibiotic therapy.

These data add further weight to the view that alternative agents to EM should be explored and thoroughly assessed for safety in pregnancy and efficacy in reducing infection-related PTB and PPROM. Further studies are also needed to explore the effect on the fetus of the sustained high concentrations of antibiotics in AF that are achieved with IA dosing.

ACKNOWLEDGMENTS

This work was supported by National Health and Medical Research Council grants PG1010315 (J.P.N.) and PG1049148 (M.W.K.), by a Women and Infant Research Foundation (Perth, Australia) Capacity Building grant (M.W.K.), and by the Microbiology and Infection Translational Research Group and the Children and Young People's Research Network as part of the Welsh Government initiative to support research (O.B.S.). Bilateral travel between Australian and United Kingdom laboratories was funded by an international exchange Royal Society grant IE130066 (O.B.S. and M.W.K.).

We acknowledge Prabha Fernandes and Cempra (Chapel Hill, NC) for the kind donation of the solithromycin (CEM-101) used in this study. We also thank Sara and Andrew Ritchie (Icon Agriculture, Darkan, Western Australia) for their expertise in providing date-mated sheep and Siemens Australia for generously donating the Rapidlab 1200 reagents and consumables used in this study.

The authors report no conflict of interest.

Footnotes

Published ahead of print 30 June 2014

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