Lucy L. Furfaro

In vitro activity of solithromycin and its metabolites, CEM-214 and N-acetyl-CEM-101, against 100 clinical Ureaplasma spp. isolates compared with azithromycin

There is a strong association between vaginal and/or amniotic fluid Ureaplasma spp. colonisation and risk of preterm birth. The novel fluoroketolide antibiotic solithromycin (CEM-101) is active against Ureaplasma spp. in vitro. Evidence from ex vivo and in vivo models suggests that, unlike most macrolide antibiotics, solithromycin readily crosses the placenta. Solithromycin metabolism varies according to species; in pregnant sheep, the bioactive metabolites CEM-214 and N-acetyl-CEM-101 (NAc-CEM-101) have been shown to accumulate in the amniotic cavity following maternal solithromycin administration, potentially contributing to its antimicrobial effects. To determine the antimicrobial activity of these metabolites against Ureaplasma spp., the effects of solithromycin, CEM-214, NAc-CEM-101 and the comparator azithromycin were tested on a collection of 100 clinical Ureaplasma spp. isolates from the UK and Australia using a modified 96-well broth microdilution method. MIC90 values observed for the combined cohort were: solithromycin, 0.125 mg/L; CEM-214, 0.5mg/L; NAc-CEM-101, 0.5mg/L; and azithromycin, 2mg/L. Solithromycin showed 34-fold greater activity against Ureaplasma spp. isolates than azithromycin, whilst CEM-214 and NAc-CEM-101 possessed ca. 22% and 17% of the activity of solithromycin, respectively, significantly greater than that of azithromycin. One bacterial isolate showed resistance to azithromycin (MIC=16 mg/L) but had a much lower MIC for solithromycin (MIC=0.25mg/L). In conclusion, the metabolites of solithromycin had reduced, but still potent, activity against 100 clinical Ureaplasma spp. isolates in vitro. This may be important in some instances such as pregnancy, however studies to determine levels of the metabolites in these settings are required.

Intrauterine Candida albicans Infection Causes Systemic Fetal Candidiasis with Progressive Cardiac Dysfunction in a Sheep Model of Early Pregnancy

Introduction: Several recent studies have identified a potential role for intrauterine Candida albicans in adverse pregnancy outcomes, including preterm birth. There is, however, a limited understanding of the impact of intrauterine candida infection on fetal well-being in early pregnancy. Using a sheep model of early pregnancy, the aims of this study were to determine (1) the ability of experimentally induced intrauterine C albicans to infect the fetus and (2) whether C albicans exposure in early pregnancy is associated with alterations in fetal cardiac function, as measured by spectral tissue Doppler imaging analysis of fetal cardiac function. Methods: Merino ewes carrying singleton pregnancies at 89 days’ gestation (term is ∼150 days) received C albicans (n = 8) via ultrasound-guided intra-amniotic injection. Saline-exposed fetuses served as controls (n = 6). Spectral tissue Doppler imaging echocardiography and amniotic fluid collection were performed at baseline and 24 and 72 hours after intrauterine C albicans injection. Fetal tissues were collected at postmortem for analysis of infection and inflammation. Results: Relative to saline control, intrauterine C albicans infection resulted in pronounced increases in amniotic fluid tumor necrosis factor α (TNF-α; P < .05) and cytokine/chemokine messenger RNA (interleukin [IL] 1β, IL-6, TNF-α, and monocyte chemoattractant protein 1; P < .05) in the fetal myocardium, lung, skin, and liver at 72 and 96 hours postinfection. Spectral tissue Doppler imaging showed diastolic dysfunction at 24 hours and severe biventricular diastolic dysfunction 72 hours postinfection. Conclusion: Intrauterine C albicans infection in a sheep model of early pregnancy causes systemic fetal candidiasis, which is associated with a robust systemic inflammatory response and progressive cardiac dysfunction detectable by spectral tissue Doppler imaging.

Applications for bacteriophage therapy during pregnancy and the perinatal period

Pregnant women and their unborn children are a population that is particularly vulnerable to bacterial infection. Physiological changes that occur during pregnancy affect the way women respond to such infections and the options that clinicians have for treatment. Antibiotics are still considered the best option for active infections and a suitable prophylaxis for prevention of potential infections, such as vaginal/rectal Streptococcus agalactiae colonization prior to birth. The effect of such antibiotic use on the developing fetus, however, is still largely unknown. Recent research has suggested that the fetal gut microbiota plays a critical role in fetal immunologic programming. Hence, even minor alterations in this microbiota may have potentially significant downstream effects. An ideal antibacterial therapeutic for administration during pregnancy would be one that is highly specific for its target, leaving the surrounding microbiota intact. This review first provides a basic overview of the challenges a clinician faces when administering therapeutics to a pregnant patient and then goes on to explore common bacterial infections in pregnancy, use of antibiotics for treatment/prevention of such infections and the consequences of such treatment for the mother and infant. With this background established, the review then explores the potential for use of bacteriophage (phage) therapy as an alternative to antibiotics during the antenatal period. Many previous reviews have highlighted the revitalization of and potential for phage therapy for treatment of a range of bacterial infections, particularly in the context of the increasing threat of widespread antibiotic resistance. However, information on the potential for the use of phage therapeutics in pregnancy is lacking. This review aims to provide a thorough overview of studies of this nature and discuss the feasibility of bacteriophage use during pregnancy to treat and/or prevent bacterial infections.

Low-dose betamethasone-acetate for fetal lung maturation in preterm sheep

BACKGROUND: Antenatal steroids are standard of care for women who are at risk of preterm delivery; however, antenatal steroid dosing and formulation have not been evaluated adequately. The standard clinical 2‐dose treatment with betamethasone‐acetate+betamethasone‐phosphate is more effective than 2 doses of betamethasone‐phosphate for the induction of lung maturation in preterm fetal sheep. We hypothesized that the slowly released betamethasone‐acetate component induces similar lung maturation to betamethasone‐phosphate+betamethasone‐acetate with decreased dose and fetal exposure. OBJECTIVE: The purpose of this study was to investigate pharmacokinetics and fetal lung maturation of antenatal betamethasone‐acetate in preterm fetal sheep. STUDY DESIGN: Groups of 10 singleton‐pregnant ewes received 1 or 2 intramuscular doses 24 hours apart of 0.25 mg/kg/dose of betamethasone‐phosphate+betamethasone‐acetate (the standard of care dose) or 1 intramuscular dose of 0.5 mg/kg, 0.25 mg/kg, or 0.125 mg/kg of betamethasone‐acetate. Fetuses were delivered 48 hours after the first injection at 122 days of gestation (80% of term) and ventilated for 30 minutes, with ventilator settings, compliance, vital signs, and blood gas measurements recorded every 10 minutes. After ventilation, we measured static lung pressure‐volume curves and sampled the lungs for messenger RNA measurements. Other groups of pregnant ewes and fetuses were catheterized and treated with intramuscular injections of betamethasone‐phosphate 0.125 mg/kg, betamethasone‐acetate 0.125 mg/kg, or betamethasone‐acetate 0.5 mg/kg. Maternal and fetal betamethasone concentrations in plasma were measured for 24 hours. RESULTS: All betamethasone‐treated groups had increased messenger RNA expression of surfactant proteins A, B, and C, ATP‐binding cassette subfamily A member 3, and aquaporin‐5 compared with control animals. Treatment with 1 dose of intramuscular betamethasone‐acetate 0.125mg/kg improved dynamic and static lung compliance, gas exchange, and ventilation efficiency similarly to the standard treatment of 2 doses of 0.25 m/kg of betamethasone‐acetate+betamethasone‐phosphate. Betamethasone‐acetate 0.125 mg/kg resulted in lower maternal and fetal peak plasma concentrations and decreased fetal exposure to betamethasone compared with betamethasone‐phosphate 0.125 mg/kg. CONCLUSION: A single dose of betamethasone‐acetate results in similar fetal lung maturation as the 2‐dose clinical formulation of betamethasone‐phosphate+betamethasone‐acetate with decreased fetal exposure to betamethasone. A lower dose of betamethasone‐acetate may be an effective alternative to induce fetal lung maturation with less risk to the fetus.

A novel one-step real-time multiplex PCR assay to detect Streptococcus agalactiae presence and serotypes Ia, Ib, and III

Streptococcus agalactiae is the leading cause of early-onset neonatal sepsis. Culture-based screening methods lack the sensitivity of molecular assays and do not indicate serotype; a potentially important virulence marker. We aimed to develop a multiplex PCR to detect S. agalactiae while simultaneously identifying serotypes Ia, Ib, and III; commonly associated with infant disease. Primers were designed to target S. agalactiae serotype-specific cps genes and the dltS gene. The assay was validated with 512 vaginal specimens from pregnant women. 112 (21.9%) were dltS positive, with 14.3%, 0.9%, and 6.3% of these identified as cps Ia, Ib, and III, respectively. Our assay is a specific and sensitive method to simultaneously detect S. agalactiae and serotypes Ia, Ib, and III in a single step. It is of high significance for clinical diagnostic applications and also provides epidemiological data on serotype, information that may be important for vaccine development and other targeted non-antibiotic therapies.

One-step simultaneous detection of Ureaplasma parvum and genotypes SV1, SV3 and SV6 from clinical samples using PlexPCR technology

Ureaplasma spp. are associated with preterm birth. In recent times, it has become apparent that Ureaplasma parvum, but not Ureaplasma urealyticum, is of most relevance. We recently demonstrated this in Australian pregnant women and using high‐resolution melt (HRM) PCR, further showed that U. parvum genotype SV6 was of particular significance. However, our assay was unable to identify multiple genotypes in the same sample, required a separate species‐level qPCR for low titre samples and was not ideal for diagnostic laboratories due to the nature of HRM PCR result interpretation. Consequently, our current study developed a novel, one‐step PlexPCR assay capable of detecting U. parvum and genotypes SV1, SV3 and SV6 in a single reaction directly from clinical samples. We then validated this using vaginal swab DNA from our Australian cohort of pregnant women. The PlexPCR was highly sensitive, detecting all targets to between 0.4 × 10−5 ng DNA (SV3) and 0.4 × 10−6 ng DNA (U. parvum, SV1 and SV6). Compared to our HRM PCR, the PlexPCR defined genotype distribution in all seven cases previously reported as ‘mixed’, and detected another eight cases where multiple genotypes (two) were present in samples previously reported as single genotypes using HRM PCR.

Bacteriophage Therapy: Clinical Trials and Regulatory Hurdles

Increasing reports of antimicrobial resistance and limited new antibiotic discoveries and development have fuelled innovation in other research fields and led to a revitalization of bacteriophage (phage) studies in the Western world. Phage therapy mainly utilizes obligately lytic phages to kill their respective bacterial hosts, while leaving human cells intact and reducing the broader impact on commensal bacteria that often results from antibiotic use. Phage therapy is rapidly evolving and has resulted in cases of life-saving therapeutic use and multiple clinical trials. However, one of the biggest challenges this antibiotic alternative faces relates to regulations and policy surrounding clinical use and implementation beyond compassionate cases. This review discusses the multi-drug resistant Gram-negative pathogens of highest critical priority and summarizes the current state-of-the-art in phage therapy targeting these organisms. It also examines phage therapy in humans in general and the approaches different countries have taken to introduce it into clinical practice and policy. We aim to highlight the rapidly advancing field of phage therapy and the challenges that lie ahead as the world shifts away from complete reliance on antibiotics.

Perinatal Streptococcus agalactiae Epidemiology and Surveillance Targets

Streptococcus agalactiae, or group B streptococcus (GBS), is a major neonatal pathogen. Recent data have elucidated the global prevalence of maternal and neonatal colonization, but gaps still remain in the epidemiology of this species. SUMMARY Streptococcus agalactiae, or group B streptococcus (GBS), is a major neonatal pathogen. Recent data have elucidated the global prevalence of maternal and neonatal colonization, but gaps still remain in the epidemiology of this species. A number of phenotypic and genotypic classifications can be used to identify the diversity of GBS strains, and some are more discriminatory than others. This review explores the main schemes used for GBS epidemiology and further details the targets for epidemiological surveillance. Current screening practices across the world provide a unique opportunity to gain detailed information on maternal colonizing strains and neonatal disease-causing strains, which is vital for monitoring and therapeutics, if sufficient detail can be extracted. Deciphering which isolates are circulating within specific populations and recording targets within invasive strains are crucial steps in monitoring the implementation of therapeutics, such as vaccines, as well as developing novel therapies against prevalent GBS strains. Having a detailed understanding of global GBS epidemiology will prove invaluable for understanding the pathogenesis of this organism and equipping future prevention strategies for success.

The efficacy of antenatal steroid therapy is dependent on the duration of low-concentration fetal exposure: evidence from a sheep model of pregnancy

BACKGROUND: Antenatal corticosteroids are among the most important and widely used interventions to improve outcomes for preterm infants. Antenatal corticosteroid dosing regimens remain unoptimized and without maternal weight‐adjusted dosing. We, and others, have hypothesized that, once a low concentration of maternofetal steroid exposure is achieved and maintained, the duration of the steroid exposure determines treatment efficacy. Using a sheep model of pregnancy, we tested the relationship among steroid dose, duration of exposure, and treatment efficacy. OBJECTIVE: The study was conducted to investigate the relative importance of duration and magnitude of fetal corticosteroid exposure to mature the preterm fetal ovine lung. STUDY DESIGN: Ewes with single fetuses at 120 days gestation received an intravenous bolus (loading dose) followed by a maintenance infusion of betamethasone phosphate to target 12‐hour fetal plasma betamethasone concentrations of (1) 20 ng/mL, (2) 10 ng/mL, or (3) 2 ng/mL. In a subsequent experiment, fetal plasma betamethasone concentrations were targeted at 2 ng/mL for 26 hours. Negative control animals received sterile saline solution. Positive control animals received 2 intramuscular injections of 0.25 mg/kg Celestone Chronodose (betamethasone phosphate + betamethasone acetate) spaced at 24 hours. Preterm lambs were delivered surgically and ventilated 48 hours after treatment commenced. Maternal and fetal plasma betamethasone concentrations were confirmed by mass spectrometry in a parallel study of chronically catheterized, corticosteroid‐treated ewes and fetuses. RESULTS: The loading and maintenance doses were achieved and maintained the desired fetal plasma betamethasone concentrations of approximately 20, 10, and 2 ng/mL for 12 hours. Compared with the 12‐hour infusion‐treated animals, lambs from the positive control (2 intramuscular doses of 0.25 mg/kg Celestone Chronodose) group had the greatest functional lung maturation (compliance, gas exchange, arterial pH) and molecular evidence of maturation (glucocorticoid receptor signaling activation), despite having maximum fetal plasma betamethasone concentrations 2.5 times lower than animals in the 20 ng/mL betamethasone infusion group. Lambs from the 12‐hour 2‐ng/mL betamethasone infusion group had little functional lung maturation. In contrast, lambs from the 26‐hour 2‐ng/mL betamethasone infusion group had functional lung maturation equivalent to lambs from the positive control group. CONCLUSION: In preterm lambs that were exposed to antenatal corticosteroids, high maternofetal plasma betamethasone concentrations did not correlate with improved lung maturation. The largest and most consistent improvements in lung maturation were in animals that were exposed to either the clinical course of Celestone Chronodose or a low‐dose betamethasone phosphate infusion to achieve a fetal plasma betamethasone concentration of approximately 2 ng/mL for 26 hours. The duration of low‐concentration maternofetal steroid exposure, not total dose or peak drug exposure, is a key determinant for antenatal corticosteroids efficacy. These findings underscore the need to develop an optimized steroid dosing regimen that may improve both the efficacy and safety of antenatal corticosteroids therapy.

Maternal Group B Streptococcus colonisation

Streptococcus agalactiae, commonly known as Group B Streptococcus (GBS), is an important neonatal pathogen known to cause sepsis, meningitis and pneumonia. Australian pregnant women undergo screening during pregnancy in an effort to eradicate GBS before delivery where transmission to the neonate can occur. Preventative treatment includes intrapartum antibiotic prophylaxis and results in widespread treatment of the 10–40% of pregnant women colonised. GBS are separated into ten different capsular polysaccharide serotypes and previous studies have suggested associations between specific serotypes anddisease. At present, however, minimal data exist on serotype distribution within Western Australian-pregnant women, information that may play an important role in future prophylactic treatment regimens. Our preliminary data, obtained from GBS isolated from vaginal swabs from 191 pregnant women, suggests that GBS serotype distributions in Western Australia are different to other parts of Australasia. In particular, compared to the eastern Australian states and New Zealand, in our cohort, serotype Ib prevalence was 7–17 times lower, II was 2–6 times greater and VI was2–12timesgreater. Inaddition, serotypeIXrepresented 6.3% of all serotypes. Understanding which serotypes are present in our population will provide valuable data for future targeted treatment regimens such as vaccinationand bacteriophage therapy. Group B Streptococcus during pregnancy Neonates are among the most vulnerable forms of life, they enter this world withminimal immune defences and are facedwith a vast array of opportunistic pathogens ready to colonise. One such organism is Streptococcus agalactiae, commonly known as Group B Streptococcus (GBS), which is responsible for morbidity and mortality in the immunocompromised, elderly and in particular, neonatal populations. GBS infection is a leading cause of sepsis and can also lead to meningitis, pneumonia, shock and even death. It is understood that transmissionof this organismcanoccur froma commensally colonised mother to her baby during birth, in utero (vertical) or alternatively through nosocomial transmission once born (horizontal). In an effort toprevent infantGBS infection, riskbased and culture-based screening of pregnantwomen followedby intrapartum antibiotic prophylaxis has been introduced in a number of countries globally. In Australia, pregnant women are screened for presence of GBS several weeks before expected delivery to determine colonisation status. If a patient is found to carry GBS, antibiotics are administered prior to delivery in an effort to eradicate the organism before the neonate is exposed.