Matthew S. Payne

Exploring Preterm Birth as a Polymicrobial Disease: An Overview of the Uterine Microbiome

Infection is a leading cause of preterm birth (PTB). A focus of many studies over the past decade has been to characterize microorganisms present in the uterine cavity and document any association with negative pregnancy outcome. A range of techniques have been used to achieve this, including microbiological culture and targeted polymerase chain reaction assays, and more recently, microbiome-level analyses involving either conserved, phylogenetically informative genes such as the bacterial 16S rRNA gene or whole shotgun metagenomic sequencing. These studies have contributed vast amounts of data toward characterization of the uterine microbiome, specifically that present in the amniotic fluid, fetal membranes, and placenta. However, an overwhelming emphasis has been placed on the bacterial microbiome, with far less data produced on the viral and fungal/yeast microbiomes. With numerous studies now referring to PTB as a polymicrobial condition, there is the need to investigate the role of viruses and fungi/yeasts in more detail and in particular, look for associations between colonization with these microorganisms and bacteria in the same samples. Although the major pathway by which microorganisms are believed to colonize the uterine cavity is vertical ascension from the vagina, numerous studies are now emerging suggesting hematogenous transfer of oral microbiota to the uterine cavity. Evidence of this has been produced in mouse models and although DNA-based evidence in humans appears convincing in some aspects, use of methodologies that only detect viable cells as opposed to lysed cells and extracellular DNA are needed to clarify this. Such techniques as RNA analyses and viability polymerase chain reaction are likely to play key roles in the clinical translation of future microbiome-based data, particularly in confined environments such as the uterus, as detection of viable cells plays a key role in diagnosis and treatment of infection.

Microbial diversity within early-stage cultured Panulirus ornatus phyllosomas.

ABSTRACT A thorough understanding of the microorganisms and pathogens associated with the larval stage of the tropical ornate rock lobster, Panulirus ornatus, is required to overcome disease outbreaks that currently block aquaculture attempts. This study used microscopy in addition to culture and molecularly based microbiological techniques to characterize the bacterial community associated with cultured, developmental stage PI to PII P. ornatus phyllosomas. Scanning electron microscopy demonstrated colonization of phyllosomas by filamentous, rod-shaped, and coccus-shaped bacteria. A clone library constructed from dead phyllosomas sampled from the larval rearing tank on day 10 was dominated by Thiothrix-affiliated sequences (56% of clones). A comparable library from live phyllosomas also contained Thiothrix-affiliated sequences, though these only represented 19% of clones within the library. Fluorescent in situ hybridization (FISH) confirmed identification of the filamentous bacteria as Thiothrix sp., being present on dead phyllosomas. FISH also identified Leucothrix sp. and Vibrio sp., as well as a range of other rod- and coccus-shaped bacteria, colonizing both live and dead phyllosomas. The development of the microbial community associated with phyllosomas was monitored through a standard larval rearing run using denaturing gradient gel electrophoresis (DGGE). Vibrio sp.-affiliated bands dominated the profiles of live animals through the rearing period and dead phyllosomas sampled on selected days. The population of Vibrio sp. associated with phyllosomas was monitored with culture-based analysis on selective media and demonstrated to increase significantly on day 7, coinciding with the beginning of the larval molt. An isolated Vibrio harveyi strain demonstrated an identical 16S rRNA sequence with retrieved DGGE and clone library sequences. Colonization of phyllosomas with filamentous bacterial species potentially hinders the ability of the animals to molt and, combined with the added stress of the molt process, likely results in reduced immune function, allowing opportunistic pathogenic Vibrio sp. to cause larval mortalities.

Does Candida and/or Staphylococcus play a role in nipple and breast pain in lactation? A cohort study in Melbourne, Australia

Objective To investigate Candida species and Staphylococcus aureus and the development of ‘nipple and breast thrush’ among breastfeeding women. Design Prospective longitudinal cohort study. Setting Two hospitals in Melbourne, Australia (one public, one private) with follow-up in the community. Participants 360 nulliparous women recruited at ≥36 weeks’ gestation from November 2009 to June 2011. Participants were followed up six times: in hospital, at home weekly until 4 weeks postpartum and by telephone at 8 weeks. Main outcome measures Case definition ‘nipple and breast thrush’: burning nipple pain and breast pain (not related to mastitis); detection of Candida spp (using culture and PCR) in the mothers vagina, nipple or breast milk or in the babys mouth; detection of S aureus in the mothers nipple or breast milk. Results Women with the case definition of nipple/breast thrush were more likely to have Candida spp in nipple/breast milk/baby oral samples (54%) compared to other women (36%, p=0.014). S aureus was common in nipple/breast milk/baby samples of women with these symptoms as well as women without these symptoms (82% vs 79%) (p=0.597). Time-to-event analysis examined predictors of nipple/breast thrush up to and including the time of data collection. Candida in nipple/breast milk/baby predicted incidence of the case definition (rate ratio (RR) 1.87 (95% CI 1.10 to 3.16, p=0.018). We do not have evidence that S aureus colonisation was a predictor of these symptoms (RR 1.53, 95% CI 0.88 to 2.64, p=0.13). Nipple damage was also a predictor of these symptoms, RR 2.30 (95% CI 1.19 to 4.43, p=0.012). In the multivariate model, with all three predictors, the RRs were very similar to the univariate RRs. This indicates that Candida and nipple damage are independent predictors of our case definition.

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

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.

Planting the seed: Origins, composition, and postnatal health significance of the fetal gastrointestinal microbiota

Abstract It has long been assumed that establishment of the fetal microbiome commences with the birthing process. However, recent studies have found bacterial DNA in umbilical cord blood, placenta, amniotic fluid, meconium, and fetal membranes in healthy normal pregnancies, leading to suggestions that the seeding of the fetal microbiome may commence in utero long before delivery. The origins of the microbiota of the fetal gastrointestinal (GI) tract have not yet been conclusively determined, although bacterial translocation from the maternal circulation, or ascension from the vagina, are both likely to be contributing pathways. Mother-to-child efflux of bacteria during pregnancy has the potential to markedly influence postnatal health, as the composition of gut microbiota determines production of important metabolites which are absorbed systemically and which modify immune function and development. Hence, the importance of understanding the colonization of the fetal GI microbiome is becoming clear, although few studies have investigated the origins, dynamics, and timing of the fetal microbiome. This is the topic of this review. By gaining a deeper understanding of the mechanisms underpinning fetal microbiome seeding, strategies may be developed to optimize fetal immune development and reduce the risk of adverse health and developmental outcomes.

Molecular Microbiological Characterization of Preterm Neonates at Risk of Bronchopulmonary Dysplasia

The role of infection in bronchopulmonary dysplasia (BPD) is unknown. We present an observational study of 55 premature infants born weighing less than 1.3 kg within two level III neonatal intensive care units. Endotracheal aspirates (ETA) and nasogastric aspirates (NGA) were studied with denaturing gradient gel electrophoresis (DGGE) profiling to elucidate the total bacterial community, and species-specific PCR was used to detect the presence of Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum. DGGE identified bacterial species in 59% of NGA and ETA samples combined. A diverse range of species were identified including several implicated in preterm labor. Species-specific PCR identified M. hominis in 25% of NGA and 11% of ETA samples. Among the 48 infants surviving up to 36 wk-postconceptual age, ordinal logistic regression showed the odds ratio for BPD or death where Ureaplasma was present/absent as 4.80 (95% CI 1.15–20.13). After adjusting for number of days ventilated, this was reduced to 2.04 (0.41–10.25). These data demonstrate how the combined use of DGGE and species-specific PCR identifies a high exposure in utero and around the time of birth to bacteria that might be causally related to preterm delivery and subsequent lung injury.

Maternal Administration of Solithromycin, a New, Potent, Broad-Spectrum Fluoroketolide Antibiotic, Achieves Fetal and Intra-Amniotic Antimicrobial Protection in a Pregnant Sheep Model

ABSTRACT Solithromycin (CEM-101) is a new antibiotic that is highly potent against Ureaplasma and Mycoplasma spp. and active against many other antibiotic-resistant organisms. We have explored the maternal-amniotic-fetal pharmacokinetics of CEM-101 in a pregnant sheep model to assess its potential for treating intrauterine and antenatal infection. Chronically catheterized pregnant ewes (n = 6 or 7) received either a single maternal intravenous (i.v.) infusion of CEM-101 (10 mg/kg of body weight), a single intra-amniotic (i.a.) injection (1.4 mg/kg of estimated fetal weight), or a combined i.v. and i.a. dose. Maternal plasma (MP), fetal plasma (FP), and amniotic fluid (AF) samples were taken via catheter at intervals of 0 to 72 h postadministration, and concentrations of solithromycin and its bioactive polar metabolites (N-acetyl [NAc]–CEM-101 and CEM-214) were determined. Following maternal i.v. infusion, peak CEM-101 concentrations in MP, FP, and AF were 1,073, 353, and 214 ng/ml, respectively, representing a maternal-to-fetal plasma transfer efficiency of 34%. A single maternal dose resulted in effective concentrations (>30 ng/ml) in MP, FP, and AF sustained for >12 h. NAc–CEM-101 and CEM-214 exhibited delayed accumulation and clearance in FP and AF, resulting in an additive antimicrobial effect (>48 h). Intra-amniotic solithromycin injection resulted in elevated (∼50 μg/ml) and sustained CEM-101 concentrations in AF and significant levels in FP, although the efficiency of amniotic-to-fetal transfer was low (∼1.5%). Combined i.v. and i.a. administration resulted in primarily additive concentrations of CEM-101 in all three compartments. Our findings suggest that CEM-101 may provide, for the first time, an effective antimicrobial approach for the prevention and treatment of intrauterine infection and early prevention of preterm birth.

Intrauterine Candida albicans infection elicits severe inflammation in fetal sheep

Background:Preventing preterm birth and subsequent adverse neonatal sequelae is among the greatest clinical challenges of our time. Recent studies suggest a role for Candida spp. in preterm birth and fetal injury, as a result of their colonization of either the vagina and/or the amniotic cavity. We hypothesized that intraamniotic Candida albicans would cause a vigorous, acute fetal inflammatory response.Methods:Sheep carrying singleton pregnancies received single intraamniotic injections of either saline (control) or 107 colony-forming units C. albicans 1 or 2 d prior to surgical delivery and euthanasia at 124 ± 2 d gestation.Results:Colonization of the amniotic cavity by C. albicans resulted in a modest inflammatory response at 1 d and florid inflammation at 2 d, characterized by fetal thrombocytopenia, lymphopenia, and significant increases of inflammatory cytokines/chemokines in the fetal membranes skin, lung, and the amniotic fluid.Conclusion:Acute colonization of the amniotic cavity by C. albicans causes severe intrauterine inflammation and fetal injury. C. albicans is a potent fetal pathogen that can contribute to adverse pregnancy outcomes.

High-Resolution Melt PCR Analysis for Genotyping of Ureaplasma parvum Isolates Directly from Clinical Samples

ABSTRACT Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.

Effects of cytokine-suppressive anti-inflammatory drugs on inflammatory activation in ex vivo human and ovine fetal membranes

Intrauterine infection and inflammation are responsible for the majority of early (<32 weeks) spontaneous preterm births (PTBs). Anti-inflammatory agents, delivered intra-amniotically together with antibiotics, may be an effective strategy for preventing PTB. In this study, the effects of four cytokine-suppressive anti-inflammatory drugs (CSAIDs: N-acetyl cysteine (NAC), SB239063, TPCA-1 and NEMO binding domain inhibitor (NBDI)) were assessed on human and ovine gestational membrane inflammation. Full-thickness membranes were collected from healthy, term, human placentas delivered by Caesarean section (n=5). Using a Transwell model, they were stimulated ex vivo with γ-irradiation-killed Escherichia coli applied to the amniotic face. Membranes from near-term, ovine placentas were stimulated in utero with lipopolysaccharide, Ureaplasma parvum or saline control and subjected to explant culture. The effects of treatment with CSAIDs or vehicle (1% DMSO) on accumulation of PGE2 and cytokines (human interleukin 6 (IL6), IL10 and TNFα; ovine IL8 (oIL8)) were assessed in conditioned media at various time points (3-20  h). In human membranes, the IKKβ inhibitor TPCA-1 (7  μM) and p38 MAPK inhibitor SB239063 (20  μM) administered to the amniotic compartment were the most effective in inhibiting accumulation of cytokines and PGE2 in the fetal compartment. NAC (10  mM) inhibited accumulation of PGE2 and IL10 only; NBDI (10  μM) had no significant effect. In addition to the fetal compartment, SB239063 also exerted consistent and significant inhibitory effects in the maternal compartment. TPCA-1 and SB239063 suppressed oIL8 production, while all CSAIDs tested suppressed ovine PGE2 production. These results support the further investigation of intra-amniotically delivered CSAIDs for the prevention of inflammation-mediated PTB.