Uncultivated Bacteria as Etiologic Agents of Intra-Amniotic Inflammation Leading to Preterm Birth

Abstract

Intra-amniotic infection and inflammation are major causes of preterm birth (PTB). However, intraamniotic inflammation is often detected in the absence of infection. This may partly be due to the culturing methods employed in hospital laboratories, which are unable to detect the uncultivated species. In this study, intra-amniotic microbial infections associated with PTB were examined by both culture and 16S rRNA-based culture-independent methods and were corroborated by the presence of intra-amniotic inflammation. Amniotic fluid (AF) specimens from 46 pregnancies complicated by PTB and 16 asymptomatic women were analyzed. No bacterial DNA was amplified in AF collected from the asymptomatic women. Among the 46 samples associated with PTB, bacterial DNA was amplified from all (16/16) of the culture-positive samples and 17% (5/30) of the culture-negative samples. In the culture-positive group, additional species were detected in more than half (9/16) of the cases by PCR and clone analysis. Altogether, approximately two-thirds of the species detected by the culture-independent methods were not isolated by culture. They included both uncultivated and difficultto-cultivate species, such as Fusobacterium nucleatum, Leptotrichia (Sneathia) spp., a Bergeyella sp., a Peptostreptococcus sp., Bacteroides spp., and a species of the order Clostridiales. To examine intra-amniotic inflammation, an AF proteomic fingerprint (mass-restricted score) was determined by surface-enhanced laser desorption ionization-time-of-flight mass spectrometry. Inflammation was detected in all five samples which were culture negative but PCR positive. Women who were PCR positive more often had elevated interleukin-6 levels in their AF, histological chorioamnionitis, and funisitis and delivered neonates with early-onset neonatal sepsis. Previously unrecognized, uncultivated, or difficult-to-cultivate species may play a key role in the initiation of PTB.

Temporal and spatial variation of the human microbiota during pregnancy

“…women with preterm labor have revealed bacterial species indigenous to the vagina, oral cavity, and gut (6)(7)(8)(9).…”

“…Evidence suggests that the most common source of invading microbes is the host microbiota. In studies of amniotic fluid from women with preterm labor and either intact or ruptured membranes, 16S ribosomal RNA (rRNA) sequences of known vaginal, gut, and oral indigenous bacterial species have been recovered in 15-50% of cases, and their relative abundances have correlated directly with markers of inflammation and inversely with time to delivery (6)(7)(8)(9). Preterm birth also is associated with bacterial vaginosis, a community-wide alteration of the vaginal microbiota (10,11) that increases the risk of preterm birth approximately twofold (12,13).…”

Does a prenatal bacterial microbiota exist?

“…17 The bacteria associated with chorioamnionitis and preterm birth by cultural methods belong to the genera Ureaplasma, Mycoplasma, Fusobacterium, Gardnerella, and Bacteroides. 13 This spectrum of cultured bacteria differs from what has been detected by PCR-based methods. 13 The reason is unclear but may again be the fastidious growth or non-culturable state of individual bacterial species or DNA contamination.…”

“…13 This spectrum of cultured bacteria differs from what has been detected by PCR-based methods. 13 The reason is unclear but may again be the fastidious growth or non-culturable state of individual bacterial species or DNA contamination. Some specialized bacteria as well as prokaryote endosymbionts have adopted lifestyles that facilitate long-term persistence within host tissue with only minor or no inflammatory response.…”

“…However, one study reported that all 16 amniotic fluid samples obtained from healthy pregnant females remained negative by PCR and culture and significant differences in the main taxa of the placental microbiome were described in three other reports. [8][9][10]13 Additionally, transient bacteremia, i.e., the presence of viable bacteria in blood is occasionally observed in the healthy host. For example, bacteria can be cultured from blood samples after dental hygiene.…”