Diversity of microbes in amniotic fluid

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Summary

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 Koch's 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.

Introduction

Intra-amniotic infection resulting from microbial invasion of the amniotic cavity (MIAC) is considered a leading etiology of preterm birth,1, 2, 3 which, in turn, is the chief cause of neonatal mortality worldwide.4 Yet our knowledge of MIAC remains incomplete, including its prevalence, optimal diagnosis, pathogenic mechanisms, and host susceptibilities. An improved understanding of the diverse microbial taxa involved in MIAC is one potentially critical step in addressing these knowledge gaps. For example, studies that characterize the microbial causes of MIAC may pinpoint species that warrant further investigations to elucidate clinically relevant traits such as virulence mechanisms, evasion of the host immune system, and antimicrobial susceptibilities. These types of insights, in conjunction with the application of improved microbial detection methods in the clinical setting may facilitate the development of new prevention, diagnosis and treatment strategies.

The primary aim of this review is to present current knowledge of the diversity of microbial taxa that invade the human amniotic cavity. Particular emphasis is placed on emerging knowledge from recent molecular studies, including evidence for causal associations with preterm birth and its adverse clinical sequelae. Related topics beyond this review's scope that are addressed elsewhere in this issue include the microbial diversity of the genital tract, and mechanisms of intrauterine infection and preterm labor.

Section snippets

Microbial detection methods for investigating MIAC: strengths and limitations

The question of whether the amniotic cavity is sterile during healthy pregnancy, and remains so throughout gestation, has been considered for at least a century.5, 6 Natural corollaries to this query are: what is the prevalence of MIAC, and what are the microbial species that cause it? Our ability to answer these questions is a function of the methods available for their investigation; as methods improve so too does our knowledge. Increasingly, molecular methods have been used to supplement

Initial investigations of MIAC: early insights from indirect evidence

In a seminal study published in 1927 of the presence of bacteria in amniotic fluid of women undergoing caesarean section, Harris and Brown found that all 28 subjects who were in labor for <6 h had negative cultures, whereas all 22 subjects who were in labor for >6 h had positive cultures.5 These findings bolstered the prevailing presumption for much of the 20th century that the amniotic cavity invariably remains sterile prior to labor onset.7, 8 Yet, some authors recognized that early onset

Fungi

Members of the genus Candida appear to be the only fungi to invade the amniotic cavity with appreciable frequency. Candida albicans is by far the predominant species recovered directly from amniotic fluid, but Candida glabrata has also been isolated from the amniotic cavity,42 and Candida parapsilosis has been reported to cause fetal infection.43 Cases of perinatal candida infections (that presumably originated as MIAC) are often associated with very early preterm births (<28 weeks) and grave

Clinical significance of the diverse microbes detected by PCR of amniotic fluid

The discovery potential of broad-range PCR is highlighted by the fact that at least six taxa in Fig. 2 are as yet uncultivated, and some have highly divergent 16S rDNA sequences from their nearest database relative (indicating that they represent previously uncharacterized species). Although a large body of compelling evidence indicates that MIAC, as detected by cultivation methods, plays a causal role in preterm birth,1, 2, 3, 60 there are legitimate reasons to question whether the detection

Evaluating causality: molecular Koch's postulates as applied to the molecular detection of microbes associated with MIAC

The gold standard criteria for establishing a causal role for microbes are the postulates of Robert Koch. Yet Koch himself realized after proposing the postulates that they could not be fulfilled for pathogens that were uncultivable by then-current methods (e.g. Vibrio cholerae, and Mycobacterium leprae).63 Because this intractable problem of uncultivable pathogens persists, an alternative causal framework based on epidemiologic guidelines suggested by Sir Austin Bradford Hill64 has been

Pathways and anatomic sources of diverse microbes that invade the amniotic cavity

Four potential routes of MIAC have been postulated: (i) ascending migration from the vagina and cervix; (ii) hematogenous dissemination followed by transplacental invasion; (iii) retrograde seeding from the peritoneal cavity via the Fallopian tubes; (iv) iatrogenic inoculation at the time of an intrauterine procedure.1

The vast majority of the bacteria and fungi that cause MIAC are thought to originate from the indigenous human microbiota (although notable exceptions exist; e.g. exogenously

Differential microbial pathogenicity and the role of microbial and host factors

A detailed discussion of pathogenic mechanisms is beyond the scope of this review. However, the pathogenesis of MIAC is likely impacted by: (i) host factors (e.g. maternal and fetal immune responses, cervical competency, barrier properties of the chorioamnion, antimicrobial properties of amniotic fluid); (ii) microbial factors (e.g. adhesion, biofilm formation, immune system evasion); and, (iii) combined factors (e.g. protective vaginal colonization). The relative contribution of specific

Is current knowledge of the microbial diversity of the amniotic cavity complete?

The microbial census of the amniotic cavity, as catalogued by broad-range PCR methods, likely remains unfinished. This shortcoming is evident from two observations. First, some microbial taxa detected in culture-based studies have yet to be detected in PCR-based studies. Second, it is likely that many microbial causes of MIAC have yet to be detected by any method. It has been estimated that 20–80% of human-associated bacterial species (depending on habitat) have yet to be isolated in pure

Conclusions

The prevalence and diversity of microbes causing MIAC is significantly greater than that indicated by culture methods, and includes as-yet uncharacterized and uncultivated taxa. The role of diverse bacteria belonging to the family Fusobacteriaceae (particularly Sneathia sanguinegens, Leptotrichia spp., and other phylogenetically related species) appears to be especially important and under-recognized. The clinical significance of a positive PCR of amniotic fluid appears to be equivalent to that

Conflict of interest statement

None declared.

Funding sources

March of Dimes Foundation.

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