Utilizing the 16S rRNA gene-based PCR and clone analysis techniques, we provide initial insight into the diversity of oral bacteria that can translocate to the murine placenta following hematogenous infection. This approach mimics transient bacteremia, which can occur during periodontal infections and dental procedures.
Bacteremia caused by bacteria from both saliva and periodontal plaque has been documented, which was why both saliva and subgingival plaque samples were tested in our study (44
). These two samples represent two distinct yet closely related floras. The concordance between the two floras has been investigated in several studies, and the correlation was found to vary from species to species (12
). Our study, likewise, confirms these findings where the saliva and dental plaque microbiota share similarities and have differences. Out of 69 different taxa identified from both samples, 10 were shared by both. As a result, the species identified in the saliva-infected placenta only partially overlap with those from the plaque-infected placenta. Even for the species that do overlap, their prevalences differ. For example, Veillonella
was one of the most prevalent bacteria in the saliva while it was not in the dental plaque. Consequently, Veillonella
was detected in five out of seven mice infected with pooled saliva but only in 1 of 10 mice infected with pooled subgingival plaque. By testing both samples, our study provides a more complete spectrum of oral species capable of transmission to placenta.
Previous studies have shown that when pregnant mice were injected with PBS, no bacteria were recovered from the placenta (29
). Thus, all bacteria detected in the placenta originated from the starting material of pooled saliva or pooled subgingival plaque. This was further confirmed by sequencing of pooled saliva and pooled subgingival plaque sample libraries, which identified the same species isolated from the mouse placentas. Studies have shown that each individual harbors approximately 266 taxa in the oral microflora (55
). Therefore, only a portion of the oral microbiome was identified, yet it appeared to be sufficient to confirm the origin of the placental infection in our study.
The bacterial prevalence in the placenta and that in the starting materials were compared. A few species that existed in high prevalence in the starting samples, such as Neisseria and Veillonella, continued to be detected in the placentas with a high prevalence, suggesting a potential dose-dependent effect. On the contrary, bacteria such as Leptotrichia colonized the placenta with decreased prevalence compared with the starting samples, suggesting a dose-independent effect. We also note the possibility of selective enrichment of several species, such as A. segnis and P. stomatis, where they may have existed in relatively low quantities in the starting pooled samples, but were identified in the placentas with increased prevalence. These observations suggest that bacteria utilize specific translocation mechanisms rather than random “diffusion” to colonize the placenta.
The number of clones analyzed from each mouse library was determined by the quality of the library and the bacterial taxa identified. For the majority of the mice, one or few predominant species were identified after sequencing just a few clones (see Tables S1 and S2 in the supplemental material). Analysis of more clones would not significantly alter the prevalence of these species. The prevalence may differ for the species that were identified only once or twice in the library. Yet, it would not affect our conclusion. In this proof-of-concept study, the answer we seek is “yes” or “no.” Even if the bacterium is identified only in the placental libraries once, it will suggest that this particular organism is capable of translocating to the placenta. Due to the intrinsic differences between humans and mice, the relative prevalence found in the mouse placenta may not be applicable to humans.
The majority of the species detected in the murine placenta have been associated with adverse pregnancy outcomes (Table ), validating the relevance of our study. Among them, F. nucleatum
is the best-recognized oral species. We have shown previously that F. nucleatum
translocates hematogenously into mouse placenta when pure cultures were used (29
). The current study demonstrates that such translocation also occurs in mixed species, better mimicking real-life situations. Furthermore, only F. nucleatum
was detected in the placenta although additional fusobacteria, such as Fusobacterium periodontium
, were present in the pooled plaque sample. This is again indicative of a species-specific translocation. This observation is consistent with those made in humans that only F. nucleatum
, not other fusobacteria, is associated with intrauterine infections.
Bacteria identified in human intrauterine infections
Some of the clones translocated to the murine placenta and those identified in intrauterine infection in humans were nonidentical but rather were closely related species of the same genus. They include Neisseria
, and Peptostreptococcus
(Table ). The discrepancy observed here may be due to the differences between humans and mice. It may also be due to the discrepancy in microbial nomenclature. It has been reported by our group and others that the nomenclature may differ based on the technology used (30
). The fact that the species identified in our study, including Leptotrichia goodfellowii
, N. flavescens
, N. subflava
, A. segnis
, Campylobacter showae
, and Capnocytophaga granulosa
, have been associated with extraoral infections involving hematogenous transmission, such as endocarditis, meningitidis, and extraoral abscesses (2
), indicates their ability to translocate to different body parts and their virulence potential.
Some species identified in the placenta exist in multiple maternal microfloras, including Campylobacter
, E. coli
, and Streptococcus
(Table ). For example, E. coli
is widely known as an enteric pathogen, but it has been also associated with stillbirth (25
). It has been considered that intra-amniotic infection with E. coli
results from the lower genital tract through an ascending route (25
). However, E. coli
can be isolated from the oral cavity even if it is not one of the most prevalent bacteria (43
). Our results indicate that the maternal-fetal infection by E. coli
and the above-mentioned species can also originate from the oral cavity as a result of bacteremia.
Some bacteria detected in the murine placenta have not been found in intrauterine infections in humans, including Erysipelothrix, Granulicatella, Microbacterium, Parvimonas (formerly known as Micromonas), Selenomonas, and the TM7 phylum. Again, this could be due to the difference between humans and mice. Interestingly, this group includes uncultivated and relatively new species, such as TM7. Thus, it is also possible that they are implicated in human intrauterine infections but have not been identified because the clinical laboratories still use routine culturing to detect microbial infections.
One very interesting observation is that many species that translocate to the murine placenta and are associated with human intrauterine infections are commensal organisms (Tables to ). For example, we identified S. mitis
, a ubiquitous oral species, in the mouse placenta. This organism, associated with various infections such as endocarditis and meningitis (14
), was recently identified in amniotic fluid from three cases of PTB (17
). Similarly, Veillonella
is also a well-recognized oral commensal species. Several reports described its association with human extraoral infections, including bacteremia, endocarditis, osteomyelitis, and meningitides, confirming its status as an opportunistic pathogen (7
). It has also been isolated from amniotic fluid accompanying PTB (47
). Therefore, our results indicate the importance of commensal oral species in intrauterine infection.
In contrast, several well-recognized periodontal pathogens, such as Porphyromonas
, and Treponema
, i.e., the “red-complex” organisms (52
), although present in the plaque sample, were not detected in the placenta. This could be due to several reasons. First, these species existed in low quantities in the plaque, placing them in a disadvantaged position in this “numbers game.” Second, the clones in the tested samples may not be the most “transmissive” isolates. Previous studies have shown that placental translocation of P. gingivalis
is strain dependent (6
How do these different bacteria colonize the mouse placenta? We have shown previously that F. nucleatum
colonizes the murine placenta by invading and crossing the endothelial lining (29
). This process requires the FadA adhesin of F. nucleatum
). Similarly, we speculate that the placental species identified in our study may possess invasive properties. Our study does not address the question as what happens after these different bacteria colonize in the mouse placenta. We have, however, demonstrated previously that after F. nucleatum
colonized the placenta, it proliferated quickly to reach a titer of 107
CFU per gram of tissue in 72 h and spread to the fetus and amniotic fluid, causing fetal death (29
). We have shown that fetal death was the result of localized TLR4-mediated inflammation following the bacterial infection. When TLR4 activation was blocked by its antagonist, F. nucleatum
still colonized the placenta to the same extent without causing fetal death (37
). It will be interesting to see if similar or different events occur with different species in the placenta.
In summary, we identified a diverse group of oral bacteria that can translocate to the mouse placenta as a result of bacteremia. These findings are consistent with the observation made in humans that various microbial species have been detected in intrauterine infection. With the exception of the two cases reported from our laboratory, the origins of most intrauterine infections were not determined. Results from the current study suggest that the oral cavity may be a previously overlooked source of intrauterine infection. Furthermore, for the first time, our study indicates the potential significance of commensal oral species in intrauterine infection. This is consistent with the previous report that many intrauterine species are of low virulence. Most of the species reported in this study have been detected in transient bacteremia in humans (4
). Previous studies have identified periodontal disease as a potential risk factor for PTB. However, intervention studies employing various periodontal therapies produced inconsistent results among pregnant women (33
). One of the most prevalent forms of periodontal disease during gestation is pregnancy-associated gingivitis, affecting at least three-quarters of the expectant mothers (3
). Gingivitis is characterized by inflamed gingiva and increased bacterial titers, including those of commensal species. These conditions may lead to frequent bacteremia, thus increasing the opportunity of hematogenous transmission. In the two cases of intrauterine infection with oral Bergeyella
or F. nucleatum
, both women showed no signs of periodontitis (a more advanced form of periodontal disease characterized by bone and attachment loss) during postpartum examinations but were suspected to have pregnancy-associated gingivitis (27
). Based on our findings, we postulate that periodontal therapies targeted at consistently reducing the total bacterial load in the mother's oral cavity may be effective in improving birth outcomes.