In this pilot study, AF samples from a total of 34 women pregnant with singletons were analyzed. The samples were obtained via transabdominal amniocentesis before the cervix was dilated >5 cm and prior to the rupture of fetal membranes. This would minimize the possibility of microbial contamination from the lower vaginal tract. PCR analysis using universal primers specific to the 16S-23S rRNA genes detected bacteria in only one patient, patient 14 (Fig. ). DNA sequence analysis of the PCR-amplified 16S rRNA gene revealed a Bergeyella strain, designated clone AF14, which shared 99.7% identity with the previously reported uncultivated oral clone AK152.
Information obtained retrospectively from the patients' medical records indicated that this was also the only patient in our study population with clinically diagnosed intrauterine infection, as indicated by a dramatically decreased glucose level and elevated WBC counts in AF. Approximately 90% of the WBC in the AF of patient 14 were neutrophils, further confirming the infectious status. Pathological analysis of her placenta following delivery showed histologic chorioamnionitis with fetal vasculitis involving the umbilical cord (funisitis) and chorionic plate, which was apparently the cause of her premature labor and delivery at 24 weeks of gestation. The acute and chronic nature of the maternal inflammatory response together with the finding of total necrosis of amnion epithelial cells is indicative of a longstanding infection of at least 3 days in duration. The fetal inflammatory response involving all three umbilical vessels and umbilical cord stroma demonstrates that the Bergeyella
sp. is capable of activating the fetal immune system, thereby increasing the risk of central nervous system and pulmonary complications (12
Little is known about the genus Bergeyella
. The type species, B. zoohelcum
, once named Weeksella zoohelcum
, is a rod-shaped, gram-negative, aerobic bacterium that is frequently associated with dog and cat bite wounds and with respiratory diseases in cats (14
). It can be found in 38 to 90% of nasal and oral fluids and gingival scrapings of dogs (38
), yet it is difficult to cultivate (40
). On the basis of the sequence of the 16S rRNA gene, oral Bergeyella
sp. clones AK152 and AF14 are clearly different from B. zoohelcum
. No oral Bergeyella
species has been cultivated to date. The difficulty in cultivation could explain why no bacteria were detected in the AF of patient 14 during route hospital testing. Although unlikely, the possibility exists that the AF of patient 14 was infected with additional species and that the AF of other patients was also infected, all at levels below the limit of detection by PCR. Since the other patients did not have clinical intrauterine infections and no bacteria were detected in their AF, PCR appears to be a reliable method for clinical diagnosis of intrauterine infections, particularly of those caused by uncultivated microorganisms. With a limit of detection of approximately 106
CFU of F. nucleatum
sp. clone AF14 likely reached a significant titer in AF. Thus, AF may be a useful supplement in media for enrichment or isolation of oral Bergeyella
Intrauterine infection with Bergeyella has never been reported before. Where could the bacteria come from? The paradigm is that intrauterine infections originate primarily from the lower genital tract via an ascending mechanism. In the case of patient 14, however, the ascending mechanism seemed questionable due to the lack of detection of Bergeyella in her vaginal swab, even with the more sensitive method of nested PCR. The lack of detection was not due to inadequate sample collection, since ample amounts of bacteria were detected in the vaginal swab, as indicated by PCR and nested PCR using universal primers (Fig. ). There is an alternative possibility that due to uneven distribution of microbes, a single sampling in the vaginal tract could have missed a particular species. Nevertheless, the same Bergeyella strain was detected in the subgingival plaque of patient 14. DNA sequence comparison of clones obtained from AF and subgingival plaque revealed a perfect match of the 16S-23S rRNA gene region, including the highly variable ITS region (Fig. ), indicating that Bergeyella sp. clone AF14 is of oral origin. Ideally, fecal samples from patient 14 should also be examined, because the gut is another major microbial habitat. However, since Bergeyella has never been reported to be associated with the gut flora, it is unlikely that it originated from the gut.
The PTB rate among the study population was higher than the national rate of 11%. This is due to the fact that women undergoing amniocentesis are usually experiencing certain complications or are under high risk and thus are more likely to deliver prematurely. However, the majority of PTBs in this study occurred late during the gestation, with a total of only six cases of preterm delivery before 30 weeks (Table ). Since intrauterine infection is more prevalent in extremely early PTB (5
), this may explain why only one case of intrauterine infection, at a rate of 3% of the study population, was identified.
The current study differs from previous ones in that bacteria identified in AF were compared with those in the vaginal tract and the oral cavity of the same patient at the DNA level (3
). Although other orally related microorganisms, such as F. nucleatum
spp., have been isolated from intrauterine infections and have been speculated to originate from the oral cavity, the presence (or absence) of these organisms in the oral cavities of the same patients was not known (4
). Oral Bergeyella
could be yet another opportunistic pathogen whose association with periodontal disease is not known but which is pathogenic when transmitted to other parts of the body such as the pregnant uterus.
How are oral bacteria transmitted from the oral cavity to the uterus? Like F. nucleatum
appears to be commonly present in the subgingival plaque (Fig. ), albeit at low levels (unpublished results; Bruce Paster, personal communication). It is unclear why it can be transmitted to the uterus in certain patients but not in others. Orogenital contact had been proposed as a possible route of transmission of oral bacteria (3
). The lack of detection of Bergeyella
in patient 14's vaginal tract did not support such a transmission (Fig. ). Thus, we speculate that the bacteria were transmitted hematogenously, as previously demonstrated in mice (18
). Is periodontal disease a prerequisite for the transmission? It is plausible that periodontal disease may facilitate the oral-utero transfer because of the increased bacterial load in the oral cavity and the altered host immune responses during disease. Although patient 14 reported gum bleeding during pregnancy, she appeared to be in good periodontal health at postpartum examination, with no significant signs of periodontal disease. Thus, the role of periodontal disease in the oral-utero transmission is unclear. It is worth pointing out that the organisms most capable of oral-utero transmission may not be those most critically implicated in periodontal disease. Host factors and the oral microflora as a whole may play important roles in the transmission process. The current study identifies a potential link between oral bacteria and preterm birth. Follow-up studies with a much larger patient population are required to address the above-mentioned questions in depth.