Pregnant mothers are susceptible to bacterial infections, which may compromise the health of mothers and offspring. Enterococcus faecalis is a ubiquitous species found in food, restaurants, and hospitals where pregnant woman frequently become exposed to this bacterium. However, the survival, distribution, translocation, and corresponding influence of E. faecalis have not been investigated during the pregnancy period, when the mother and fetus are susceptible to bacterial infection. In this study, a fluorescing E. faecalis strain was used to track the fate of the bacterium in pregnant mice. Orally administered E. faecalis were found to survive and disseminate to all regions of the intestinal tract. It also altered the bacterial community structure by significantly decreasing the diversity of Lactobacillus species, impairing the normal structure and function of the intestinal barrier, which may contribute to the bacterial translocation into the blood, spleen, placenta, and fetus. This may affect fetal and placental growth and development.
Enterococcus faecalis has been widely isolated from the environment (Rathnayake et al., 2011), food (Giraffa, 2006), and digestive tracts (Nueno-Palop & Narbad, 2011). In the past, E. faecalis was considered a harmless bacterium. Several strains have even been used as probiotics to promote a positive gut environment (Franz et al., 2011). However, this consensus has been challenged (Arias & Murray, 2012). Research has shown E. faecalis is a common pathogen in nosocomial infections, and may disrupt the intestinal microbial balance and induce inflammation (Balish & Warner, 2002; Kim et al., 2007; Lupp et al., 2007).
A pregnant individual is susceptible to pathogen infection, which may compromise the health of both mother and offspring, such as cytomegalovirus (Scott et al., 2012), Trypanosoma cruzi (Duaso et al., 2011), Salmonella (Chattopadhyay et al., 2010), and Listeria monocytogenes (Lamont et al., 2011). Although E. faecalis has been clinically detected in breast milk, placenta, amniotic fluid, umbilical cord blood, and meconium (Martı́n et al., 2004; Jiménez et al., 2008), its effect on pregnant individuals has rarely been a concern. Previous reports mentioned that E. faecalis is able to cross the intestinal barrier and subsequently cause disease (Wells et al., 1990; Zeng et al., 2004; Steck et al., 2011). However, the survival, distribution, translocation, and corresponding influence of E. faecalis have not been investigated during the pregnancy period, when the mother and fetus are susceptible to bacterial infection. The goal of this study is to investigate the effect of E. faecalis on pregnant mice.
Materials and methods
Bacterial strain and culture condition
Enterococcus faecalis OG1RF with plasmid pMV158GFP (tetracycline resistance, TcR) was provided by Manuel Espinosa (Nieto & Espinosa, 2003). The strain was cultured in TSB medium containing 4 μg mL−1 tetracycline. The cells were harvested and reconstituted to 109 CFU mL−1 before use.
Animals were cared for following institutional animal care committee guidelines (NIH, Publication No. 85–23, 1996). All procedures were conducted in compliance with protocols provided and approved by the Animal Care Review Committee (approval number 0064257), Nanchang University, Jiangxi, China. Twelve-week-old KM pregnant mice were bred in the experimental animal center of Nanchang University. The mice were housed in cages at 25 °C with a 12 h light/dark cycle, and fed with water and mice laboratory diet daily. All mice were sacrificed using CO2 at the end of experiment.
Enterococcus faecalis oral infection
Pregnant mice were randomly assigned to the test or the control group at the first day of pregnancy. The control group was orally administered phosphate-buffered saline (PBS) and the test group received 100 μL bacterial suspension once a day for 19 days (the gestation period of mice is 21 days). A day before the predicted delivery date, all mice were sacrificed and surface-sterilized with 75% ethanol. Blood was obtained by eyeball enucleation. The animals were dissected to collect the respective organs including the stomach, intestine, heart, brain, liver, spleen, kidney, placenta, and fetus.
Detection of E. faecalis in pregnant mice
All tissue samples were homogenized in PBS, serially diluted and spread on TSB TcR agar plate. All plates were incubated at 37 °C for 24–48 h until the green fluorescent colonies formed. Both fluorescence and tetracycline resistance were used as the detection marker for E. faecalis OG1RF:pMV158GFP. Preliminary experimental results revealed that other tetracycline-resistant bacteria were also present in the digestive tracts of pregnant mice.
Total genomic DNA was extracted following a bead-beating method. PCR was performed using a Taq DNA polymerase kit (CoWin Biotech Co., Ltd, Beijing, China). All primers used are listed in Supporting Information, Table S1. The PCR conditions were as follows: 94 °C for 5 min; 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; followed 72 °C for 10 min. The PCR products were analyzed with 1.5% agarose gels.
Denaturing gradient gel electrophoresis (DGGE) analysis of the PCR products was performed with a DCode™ Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA). The DGGE patterns were normalized and analyzed with bionumeric software version 2.0 (Applied Maths, London, UK). During processing, different lanes were defined, the background was subtracted, differences in the intensity of lanes were compensated during normalization, and the the correlation matrix was calculated. Clustering was done with Pearson correlation and the UPGMA method. Diversity indices were also calculated: richness (S) was defined as the number of bands in one lane, the Shannon–Wiener index (H′) was calculated as , where Pi is the importance probability of the bands in a lane, calculated from Ni/N, where Ni is the peak height of a band and N is the sum of all peak heights, Evenness (E) was calculated from , where (Yu & Morrison, 2004). Simpson's index (D) was calculated as (Menezes Bento et al., 2005). Finally, each band of interest was excised from the gels, then transferred into a 1.5-mL tube containing 30 μL TE buffer and incubated overnight at 4 °C to allow the DNA to diffuse into the buffer. The collected DNA solutions were subsequently used for sequencing. The sequences were compared using blast-n provided by NCBI blast (http://www.ncbi.nlm.nih.gov).
Histopathology and immunohistochemistry
Small pieces of ileum, caecum and colon were excised and fixed in 10% formalin. These samples were dehydrated and embedded in paraffin wax. Subsequently, 5-μm-thick tissue sections were cut. These sections were transferred onto clean slides, dried overnight and stored at room temperature until use. The mounted tissue sections were stained with hematoxylin and eosin for histologic examination. For pathological scoring, six fields per sample were examined and scored according to the method described by Yang et al. (2013).
Antigen was retrieved in 0.01 M saline-sodium citrate solution by microwave irradiation. The 5-μm-thick sections were stained and visualized using a Dako REAL EnVision™ Detection System, Peroxidase/DAB, Rabbit/Mouse (K5007, Dako, Glostrup, Denmark). Anti-E-cadherin (sc-8426) 1 : 200 (Santa Cruz Biotechnology, Santa Cruz, CA) was used as primary antibody; biotinylated goat anti-rabbit IgG served as secondary antibody, and streptavidin-horseradish peroxidase provided in the kit was used for signal transduction. Subsequently, the sections were photographed with a microscope (Nikon Corp., Tokyo, Japan) and the results were analyzed with Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MA). Mean optical density (MOD) (integrated optical density/area) was used to calculate the levels of E-cadherin expression in the tissues.
Unless otherwise specified, all experiments were repeated in triplicate. All data were analyzed with Sigma Plot 11.0 (Systat Software, San Jose, CA) and spss version 16.0 (SPSS Inc., Chicago, IL).
Distribution and translocation of viable fluorescent E. faecalis in pregnant mice
As shown in Fig. 1a, the viable fluorescent E. faecalis in different regions of the digestive tract varied greatly. No viable fluorescing E. faecalis was detected from the stomach, suggesting that E. faecalis OG1RF was unable to colonize this region. However, the numbers of fluorescing E. faecalis from the large intestine were ≥ 5.6 ± 0.4 log10 CFU g−1. The cell counts in the caecum ranged from 5.6 ± 0.4 to 6.8 ± 0.2 log10 CFU g−1. The cell counts in the small intestine were variable. In three of five pregnant mice, cell counts ranged from 3.8 ± 0.2 to 4.0 ± 0.4 log10 CFU g−1, but the target bacteria were not found in the small intestines of the other two mice. In general, the survey indicated that orally administered, E. faecalis OG1RF could survive and distribute through whole intestinal tracts, especially the caecum.
Figure 1b showed that fluorescing E. faecalis were found in various tissues, evidence of the ability of the orally administered bacteria to translocate. In general, no viable fluorescing E. faecalis was found in the brain, heart, liver or kidney. However, it was detected in the blood, spleen, placenta, and fetus. The bacteria in the blood ranged from 2.1 ± 0.1 to 2.3 ± 0.3 log10 CFU mL−1, and in the spleen from 2.4 ± 0.4 to 2.7 ± 0.4 log10 CFU g−1. In addition, in two mice placentas, relatively low counts of fluorescing E. faecalis (≤ 1.9 ± 0.2 Log10 CFU g−1) was detected. Moreover, in one mouse, a few viable fluorescent E. faecalis were observed in its fetus.
Effects of orally administered E. faecalis on bacterial community diversity at the caecum
Changes in the indices for the normal microbiota and Lactobacillus species were determined by PCR-DGGE. Figure S1 shows that the similarity index between the control and treated group was 0.50–0.70 and 0.28–0.40, respectively. In Fig. 2b, the similarity indices were 0.45–0.70 and 0.24–0.49 for the Lactobacillus species. Generally, these results indicate that the bacterial community in the caecum is relatively stable but changed dramatically upon oral administration of E. faecalis.
Based on PCR-DGGE, the dominant bacteria in the caecum of the control pregnant mice were Proteobacterium (vc-a), uncultured bacterium (vc-b), Lachnospiraceae (vc-c), Bacteroidales (vc-d), uncultured bacterium (vc-e), and Gammaproteobacterium (vc-f) as seen in Table 1. However, after oral infection with E. faecalis, the Lachnospiraceae (vc-g) showed up as dominant in three treated mice and the uncultured bacterium (vc-h) become the dominant bacteria in the other three treated mice; vc-d corresponding to Bacteroidales either diminished or disappeared (Table 1). In addition, no significant difference was detected in the community structure diversity of the normal microbiota (Table S2). The dominant Lactobacillus in the control pregnant mice were Lactobacillus johnsonii (lc-a) and Lactobacillus sp. (lc-b, c, and d). In the test animals, L. johnsonii (lc-e), Lactobacillus reuteri (lc-f) and Lactobacillus gasseri (lc-g) were recorded as the dominant species (Table 1). Moreover, we found the Lactobacillus structure diversity (Richness and Shannon–Wiener index) of the test group was significantly decreased (Table S2). Hence, the results support the significant effect of orally administered E. faecalis on the diversity of caecum.
Table 1. Sequencing results of selected DGGE bands in Fig. S1 and Fig. 2
Effects of orally administered E. faecalis on intestinal structure and function
The representative histological photomicrographs of the ileum, caecum, and colon are shown in Fig. 3a–c, respectively. Histological analysis showed that several lesions (crypt damages, goblet cell reduction, and epithelial erosions) were detected in the epithelium of treated mice. These results are indicative of possible damage resulting from the oral administration of E. faecalis in pregnant mice.
The representative photomicrographs of the ileum, caecum, and colon that were immunostained for E-cadherin are shown in Fig. 4a–c, respectively. The MOD of tissue sections was used to quantify the expression of E-cadherin in the intestine (Fig. 4d). Results indicated that the MOD of treated ileum (75.4 ± 11.0) was significantly less than the control (93.3 ± 6.3). Similarly, as compared with control (59.1 ± 7.4), the MOD of treated caecum (36.7 ± 12.9) was significantly decreased. The MOD of control colon was 54.6 ± 9.2, significantly higher than the MOD of treated colon (37.6 ± 7.8). These results indicated that the presence of E-cadherin was significantly reduced in the intestine of pregnant mice after oral infection with E. faecalis. Hence, it may be inferred that oral administration of E. faecalis contributed to the impairment of the epithelial barrier function of the pregnant mothers.
Effects of orally administered E. faecalis on fetal and placental growth of mice
The fetal size was dramatically reduced when pregnant mice were administered E. faecalis orally (Fig. 5a). The fetal weight was only 1.22 ± 0.13 g in the treated group, as compared with 1.63 ± 0.30 g in the control, a 25.6% reduction (Fig. 5a). Placental weights also decreased from 0.13 ± 0.02 g in the control group to 0.11 ± 0.01 g in the in the E. faecalis -treated group, with a 15.4% reduction (Fig. 5b). Hence, the results indicate that oral administration of E. faecalis may stunt fetal and placental growth and development.
Intestinal survival and colonization is the critical prerequisite for both food-borne pathogens and commensal bacteria to persist and function in the gut (Shi & Walker, 2004). Understanding the fate of bacteria in the digestive tract may elucidate where, how, and what role the bacterium plays. In this study, we investigated the distribution of E. faecalis in various sections of pregnant mice (Fig. 1a). Our results show that E. faecalis was able to survive in the intestinal tract and persist among the gut microbiota of pregnant mice.
The effects of intestinal survival and distribution after oral administration were studied using PCR-DGGE. PCR-DGGE is an effective fingerprinting technique to assess microbial community distribution and dynamics (Hawkins & Purdy, 2007). According to the results, oral administration of E. faecalis altered the bacterial community distribution. Notably, the diversity of Lactobacillus species (indicated by the Richness and Shannon–Wiener Index) was significantly decreased in mice treated with E. faecalis (Table S2). This alteration may reduce intestinal host defense and lead to the destruction of the normal structure and function of the intestinal tract (Round & Mazmanian, 2009; Sekirov et al., 2010).
The effect of E. faecalis on the intestines of pregnant mice was also investigated. The results of the histological studies on the intestine of pregnant mice that were administered E. faecalis orally showed pathological changes (Fig. 3). In the past, several reports revealed that E. faecalis cause colonic inflammation and chronic colitis in interleukin-10−/− mice but not in wild-type mice (Kim et al., 2005). Moreover, E. faecalis was shown to affect the expression of E-cadherin through cell–cell adhesion and epithelial barrier function (Steck et al., 2011). Similarly, the results suggested that the expression of E-cadherin in all intestinal regions was significantly reduced in comparison with the control (Fig. 4). This implies that the presence of E. faecalis impairs the integrity of the epithelial barrier during pregnancy in mice.
Based on the results gathered, orally administered E. faecalis was translocated into the blood and spleen (Fig. 1b). In addition, the E. faecalis induced alterations of bacterial community population and impaired the normal intestinal structure and function, which enabled E. faecalis to translocate into the bloodstream and spleen. Bacterial translocation is recognized as an important factor contributing to the occurrence and development of diseases (Van Leeuwen et al., 1994). In 1990, Wells et al. observed the translocation of E. faecalis across the intestinal tract of mice. Zeng et al. (2004, 2005) compared translocation ability among several E. faecalis strains and demonstrated that epa and gelatinase are important for its translocation. Tsuda and Shigematsu noted that thermally injured mice are susceptible to E. faecalis translocation, which subsequently causes sepsis (Tsuda et al., 2008; Shigematsu et al., 2012). This study showed that orally administered E. faecalis survived in the gut, translocated and caused bacteremia in pregnant mice.
Furthermore, in addition to causing maternal disorders, translocation of E. faecalis, even at relatively low frequencies, was observed in maternal placentas and fetuses (Fig. 1b). These have been suggested to have an impact on the growth and development of the fetus (Rezeberga et al., 2008). In line with our speculations, the results showed that maternal E. faecalis treatment reduced fetal and placental weight (Fig. 5).
In summary, our results showed that orally administered E. faecalis can survive in the intestine of pregnant mice. These E. faecalis may induce the alteration of the inherent bacterial community structure, especially the diversity of Lactobacillus species. Additionally, the E. faecalis bacterial–host interaction can impair the integrity of the intestinal barrier, which may have contributed to the translocation of E. faecalis into the blood, spleen, placenta, and fetus. This may stunt fetal and placental growth and development.
This research project was supported by the National Natural Science Foundation of China (NSF31170091, 31000048), Academic and Technical Leaders Training Program for Major Subjects of Jiangxi Province (2009), and the Research Program of the State Key Laboratory of Food Science and Technology of Nanchang University (SKLF-TS-200916, SKLF-MB-201002).