Daniel B. DiGiulio

Microbial Prevalence, Diversity and Abundance in Amniotic Fluid During Preterm Labor: A Molecular and Culture-Based Investigation

Background Preterm delivery causes substantial neonatal mortality and morbidity. Unrecognized intra-amniotic infections caused by cultivation-resistant microbes may play a role. Molecular methods can detect, characterize and quantify microbes independently of traditional culture techniques. However, molecular studies that define the diversity and abundance of microbes invading the amniotic cavity, and evaluate their clinical significance within a causal framework, are lacking. Methods and Findings In parallel with culture, we used broad-range end-point and real-time PCR assays to amplify, identify and quantify ribosomal DNA (rDNA) of bacteria, fungi and archaea from amniotic fluid of 166 women in preterm labor with intact membranes. We sequenced up to 24 rRNA clones per positive specimen and assigned taxonomic designations to approximately the species level. Microbial prevalence, diversity and abundance were correlated with host inflammation and with gestational and neonatal outcomes. Study subjects who delivered at term served as controls. The combined use of molecular and culture methods revealed a greater prevalence (15% of subjects) and diversity (18 taxa) of microbes in amniotic fluid than did culture alone (9.6% of subjects; 11 taxa). The taxa detected only by PCR included a related group of fastidious bacteria, comprised of Sneathia sanguinegens, Leptotrichia amnionii and an unassigned, uncultivated, and previously-uncharacterized bacterium; one or more members of this group were detected in 25% of positive specimens. A positive PCR was associated with histologic chorioamnionitis (adjusted odds ratio [OR] 20; 95% CI, 2.4 to 172), and funisitis (adjusted OR 18; 95% CI, 3.1 to 99). The positive predictive value of PCR for preterm delivery was 100 percent. A temporal association between a positive PCR and delivery was supported by a shortened amniocentesis-to-delivery interval (adjusted hazard ratio 4.6; 95% CI, 2.2 to 9.5). A dose-response association was demonstrated between bacterial rDNA abundance and gestational age at delivery (r2 = 0.42; P<0.002). Conclusions The amniotic cavity of women in preterm labor harbors DNA from a greater diversity of microbes than previously suspected, including as-yet uncultivated, previously-uncharacterized taxa. The strength, temporality and gradient with which these microbial sequence types are associated with preterm delivery support a causal relationship.

Prevalence and diversity of microbes in the amniotic fluid, the fetal inflammatory response, and pregnancy outcome in women with preterm pre-labor rupture of membranes.

Citation DiGiulio DB, Romero R, Kusanovic JP, Gómez R, Kim CJ, Seok K, Gotsch F, Mazaki‐Tovi S, Vaisbuch E, Sanders K, Bik EM, Chaiworapongsa T, Oyarzún E, Relman DA. Prevalence and diversity of microbes in the amniotic fluid, the fetal inflammatory response, and pregnancy outcome in women with preterm pre‐labor rupture of membranes. Am J Reprod Immunol 2010; 64: 38–57

Temporal and spatial variation of the human microbiota during pregnancy.

Significance The human indigenous microbial communities (microbiota) play critical roles in health and may be especially important for mother and fetus during pregnancy. Using a case-control cohort of 40 women, we characterized weekly variation in the vaginal, gut, and oral microbiota during and after pregnancy. Microbiota membership remained relatively stable at each body site during pregnancy. An altered vaginal microbial community was associated with preterm birth; this finding was corroborated by an analysis of samples from an additional cohort of nine women. We also discovered an abrupt change in the vaginal microbiota at delivery that persisted in some cases for at least 1 y. Our findings suggest that pregnancy outcomes might be predicted by features of the microbiota early in gestation. Despite the critical role of the human microbiota in health, our understanding of microbiota compositional dynamics during and after pregnancy is incomplete. We conducted a case-control study of 49 pregnant women, 15 of whom delivered preterm. From 40 of these women, we analyzed bacterial taxonomic composition of 3,767 specimens collected prospectively and weekly during gestation and monthly after delivery from the vagina, distal gut, saliva, and tooth/gum. Linear mixed-effects modeling, medoid-based clustering, and Markov chain modeling were used to analyze community temporal trends, community structure, and vaginal community state transitions. Microbiota community taxonomic composition and diversity remained remarkably stable at all four body sites during pregnancy (P > 0.05 for trends over time). Prevalence of a Lactobacillus-poor vaginal community state type (CST 4) was inversely correlated with gestational age at delivery (P = 0.0039). Risk for preterm birth was more pronounced for subjects with CST 4 accompanied by elevated Gardnerella or Ureaplasma abundances. This finding was validated with a set of 246 vaginal specimens from nine women (four of whom delivered preterm). Most women experienced a postdelivery disturbance in the vaginal community characterized by a decrease in Lactobacillus species and an increase in diverse anaerobes such as Peptoniphilus, Prevotella, and Anaerococcus species. This disturbance was unrelated to gestational age at delivery and persisted for up to 1 y. These findings have important implications for predicting premature labor, a major global health problem, and for understanding the potential impact of a persistent, altered postpartum microbiota on maternal health, including outcomes of pregnancies following short interpregnancy intervals.

Diversity of microbes in amniotic fluid

Recent polymerase chain reaction (PCR)-based studies estimate the prevalence of microbial invasion of the amniotic cavity (MIAC) to be ≥30-50% higher than that detected by cultivation-based methods. Some species that have been long implicated in causing MIAC remain among the common invaders (e.g. Ureaplasma spp., Mycoplasma spp., Fusobacterium spp. Streptococcus spp., Bacteroides spp. and Prevotella spp.). Yet we now know from studies based on PCR of the 16S ribosomal DNA that cultivation-resistant anaerobes belonging to the family Fusobacteriaceae (particularly Sneathia sanguinegens, and Leptotrichia spp.) are also commonly found in amniotic fluid. Other diverse microbes detected by PCR of amniotic fluid include as-yet uncultivated and uncharacterized species. The presence of some microbial taxa is associated with specific host factors (e.g. Candida spp. and an indwelling intrauterine device). It appears that MIAC is polymicrobial in 24-67% of cases, but the potential role of pathogen synergy is poorly understood. A causal relationship between diverse microbes, as detected by PCR, and preterm birth is supported by types of association (e.g. space, time and dose) proposed as alternatives to Kochs postulates for inferring causality from molecular findings. The microbial census of the amniotic cavity remains unfinished. A more complete understanding may inform future research directions leading to improved strategies for preventing, diagnosing and treating MIAC.

Microbial invasion of the amniotic cavity in preeclampsia as assessed by cultivation and sequence-based methods.

Abstract Objective: Infection has been implicated in the pathogenesis of preeclampsia, yet the association between microbial invasion of the amniotic cavity (MIAC) and preeclampsia has not been determined. The aim of this study was to determine the prevalence, and microbial diversity associated with MIAC, as well as the nature of the host response to MIAC in patients with preeclampsia. Method of study: Amniotic fluid (AF) from 62 subjects with preeclampsia, not in labor, was analyzed with both cultivation and molecular methods. Broad-range and group-specific PCR assays targeting small subunit ribosomal DNA, or other gene sequences, from bacteria, fungi and archaea were used. Results were correlated with measurements of host inflammatory response, including AF white blood cell count and AF concentrations of glucose, interleukin-6 (IL-6) and MMP-8. Results: 1) The rate of MIAC in preeclampsia was 1.6% (1/62) based on cultivation techniques, 8% (5/62) based on PCR, and 9.6% (6/62) based on the combined results of both methods; 2) among the six patients diagnosed with MIAC, three had a positive PCR for Sneathia/Leptotrichia spp.; and 3) patients with MIAC were more likely to have evidence of an inflammatory response in the amniotic cavity than those without MIAC, as determined by a higher median AF IL-6 [1.65 ng/mL interquartile range (IQR): 0.35–4.62 vs. 0.22 ng/mL IQR: 0.12–0.51; P=0.002). Conclusion: The prevalence of MIAC in preeclampsia is low, suggesting that intra-amniotic infection plays only a limited role in preeclampsia. However, the unexpectedly high number of positive AF specimens for Sneathia/Leptotrichia warrants further investigation.

Scedosporium apiospermum Soft Tissue Infection Successfully Treated with Voriconazole: Potential Pitfalls in the Transition from Intravenous to Oral Therapy

ABSTRACT An immunocompromised patient with an invasive soft tissue infection due to Scedosporium apiospermum was successfully treated with voriconazole and surgical debridement. After transition from intravenous to oral therapy, successive adjustments of the oral dose were required to achieve complete resolution. For soft tissue infections due to molds characterized by thin, septate hyphae branching at acute angles, voriconazole should be considered a first-line antifungal agent. The potential usefulness of plasma voriconazole levels for guiding optimal therapy should be investigated.

Replication and refinement of a vaginal microbial signature of preterm birth in two racially distinct cohorts of US women

Significance Premature birth (PTB) is a major global public health burden. Previous studies have suggested an association between altered vaginal microbiota composition and PTB, although findings across studies have been inconsistent. To address these inconsistencies, improve upon our previous signature, and better understand the vaginal microbiota’s role in PTB, we conducted a case-control study in two cohorts of pregnant women: one predominantly Caucasian at low risk of PTB, the second predominantly African American at high risk. With the results, we were able to replicate our signature in the first cohort and refine our signature of PTB for both cohorts. Our findings elucidate the ecology of the vaginal microbiota and advance our ability to predict and understand the causes of PTB. Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality. Previous studies have suggested that the maternal vaginal microbiota contributes to the pathophysiology of PTB, but conflicting results in recent years have raised doubts. We conducted a study of PTB compared with term birth in two cohorts of pregnant women: one predominantly Caucasian (n = 39) at low risk for PTB, the second predominantly African American and at high-risk (n = 96). We profiled the taxonomic composition of 2,179 vaginal swabs collected prospectively and weekly during gestation using 16S rRNA gene sequencing. Previously proposed associations between PTB and lower Lactobacillus and higher Gardnerella abundances replicated in the low-risk cohort, but not in the high-risk cohort. High-resolution bioinformatics enabled taxonomic assignment to the species and subspecies levels, revealing that Lactobacillus crispatus was associated with low risk of PTB in both cohorts, while Lactobacillus iners was not, and that a subspecies clade of Gardnerella vaginalis explained the genus association with PTB. Patterns of cooccurrence between L. crispatus and Gardnerella were highly exclusive, while Gardnerella and L. iners often coexisted at high frequencies. We argue that the vaginal microbiota is better represented by the quantitative frequencies of these key taxa than by classifying communities into five community state types. Our findings extend and corroborate the association between the vaginal microbiota and PTB, demonstrate the benefits of high-resolution statistical bioinformatics in clinical microbiome studies, and suggest that previous conflicting results may reflect the different risk profile of women of black race.

Molecular mycological diagnosis and correct antimycotic treatments.

In a recent report, Schaenman and colleagues (5) describe a case of a Scedosporium apiospermum soft tissue infection in an immunocompromised patient successfully treated with voriconazole. The article focuses on one of the hottest topics in current medical mycology, the emergence of antimycotic-resistant fungal isolates (3). Indeed, Scedosporium apiospermum is, also in our direct experience, one of the emerging fungal pathogens frequently endowed with resistance against drugs used as first-line agents (i.e., amphotericin B and fluconazole) (2). Therefore, we agree to the general message of the paper regarding the need of a prompt and well designed antifungal therapy. However, we would like to address a major point on how this could be achieved. In fact, we disagree that presumptive fungal identification based on aspecific morphological aspects is sufficient to take into account a new drug such as voriconazole as a first-line agent in the management of fungal infections. As admitted by the authors and clearly shown in Fig. 2 of their case report, many fungal genera feature morphological characteristics difficult to discriminate and the identification is not straightforward, especially if specific structures are not usually evident, as may be the case upon direct examination of clinical samples. A clinician making the same assumption as the authors might feel free to treat critically ill patients with voriconazole in the majority of cases, thus putting the whole community at risk for the emergence of new resistances to this valuable drug, as has already and inevitably happened for narrow-spectrum triazoles. Moreover it is still far from being proven that the toxicity profile of the new extended-spectrum triazoles is really safer than that of narrow-spectrum drugs, with severe side effects reported in up to 10% of patients receiving voriconazole (1). We think that a rapid and precise identification, at least at the genus level, is crucial for the prescription of a well designed empirical therapy, but we are convinced that it should be based on objective data. Recently, we addressed the diagnosis of mycotic keratitis using, in parallel with cultural methodologies, a molecular approach based on direct amplification from the biological sample and sequencing, by means of universal fungal primers (4, 6), of genus- and species-specific targets on the fungal genome. In our opinion this molecular approach allowing unequivocal identification of a fungal pathogen, at least at the genus level, in only one day is, together with a more thorough understanding of mechanisms of drug resistance, a real improvement of the conventional mycological diagnosis and represents a correct answer to the clinical questions posed by the availability of multiple classes of antifungal agents.

Majority Rules? Tallying the Microbial Census in an Abscess by Means of Molecular Methods

Received 15 January 2009; accepted 17 January 2009; electronically published 31 March 2009. Reprints or correspondence: Dr. David A. Relman, VAPAHCS 154T, 3801 Miranda Ave., Palo Alto, CA 94304 (relman@ stanford.edu). Clinical Infectious Diseases 2009; 48:1179–81 2009 by the Infectious Diseases Society of America. All rights reserved. 1058-4838/2009/4809-0002

Prematurity and perinatal antibiotics: a tale of two factors influencing development of the neonatal gut microbiota.

15.00 DOI: 10.1086/597579 It is a scenario familiar to virtually every infectious disease clinician: a patient exhibits unambiguous signs of infection, yet the results of cultures of appropriate clinical specimens remain negative. This hinders our ability to answer an important clinical question: “who”—in the microbial sense—is present at the disease site? Absent this information, empirical therapy is guided by the answer to a second pressing question: “who” could potentially be present? To answer this query for a given infectious syndrome requires knowledge of the diversity of microbial taxa that contribute to pathology at an anatomic site of interest. At the same time, we must avoid jumping to conclusions of “guilt” based solely on findings of colocalization with pathology; not all microbial species found in an abscess, in particular, are critical to—or even involved in—pathogenesis, nor do they necessarily require targeted therapy. In this issue, Al Masalma et al. [1] provide the most revealing glimpse to date of bacterial diversity within brain abscesses. Brain abscesses continue to be an important clinical problem: the mortality rate among immunocompetent patients remains as high as 4%–12% in the current era of CT [2, 3], and the incidence of neurologic sequelae is 120% [2]. However, despite the seriousness of this syndrome, the clinical benefit of identifying the lessabundant microbial constituents in abscesses remains unproven. We know from animal models, however, that polymicrobial infections are often complex, dynamic, and interactive processes. For example, avirulent [4, 5] and even beneficial [6] bacterial strains may synergistically enhance pathogenicity as part of a microbial community. Pathogen synergy is orchestrated by elaborate interactions [6] between microbes possessing diverse capabilities, including, among others, biofilm formation [7], phagocytosis inhibition [8], redox potential reduction [9], toxin production [10], nutrient provision [9], and transcriptional modulation of virulence factors in colocated species [6]. Furthermore, marked population shifts in absolute and relative abundance of diverse microbial species may occur during the evolution of a polymicrobial infection [11]. Although the role of numerically nondominant microbial taxa in initiating, maintaining, and exacerbating brain abscesses has been little studied, it makes sense to begin by first documenting and enumerating potential “players,” including any statistically significant co-occurrence of specific microbial groups. In their study, Al Masalma et al. [1] used a PCRand sequence-based approach that exploits 16S ribosomal DNA (rDNA) to profile bacterial diversity. The rationale for this cultivation-independent strategy is clear. It has long been recognized that many microbes visualized by microscopy cannot be cultivated [12]. Despite advances in cultivation technology, 99% of the microbial species residing in various habitats cannot be recovered by available techniques, a phenomenon termed the “great plate count anomaly” [13]. In addition, attempts to isolate even cultivable pathogens may fail. This is especially true for fastidious microbial groups, such as microaerophiles and strict anaerobes, members of which are often found in brain abscesses [2, 3, 14]. Finally, the yield from microbial cultivation may be compromised when antibiotic administration precedes the sampling of brain abscess material [3]. To overcome shortcomings of culture, Al Masalma and colleagues amplified bacterial 16S rDNA sequences from intra-operatively collected abscess specimens [1] by means of broadrange PCR primers. These primers target sites of known sequence conservation within the 16S rRNA gene while enabling