During the 1660s–1670s, English botanist Robert Hooke (1635–1702) observed cork, bone, and plants with microscope magnification of 30 times, then put forward the “cell” concept (1665). At the same time, the Dutch scientist Anton van Leeuwenhoek (1632–1723) improved microscope magnification up to 300 times. This helped him to observe living creatures swimming in a drop of pond water, which he named “animalcules” (1676). These two isolated breakthroughs of Hooke and van Leeuwenhoek gave birth to modern cytology, histology, histopathology, and to modern microbiology, respectively. Hooke concentrated on revealing the basic composition of animals and plants, while Leeuwenhoek concentrated on the observation of free-living microorganisms in nature, including red blood cells and spermatozoa. In the following decades, with the work of Marcello Malpighi (1628–1694), Nehemiah Grew (1641–1712), Theodore Schwann (1810–1882), Mattias Jacob Schleiden (1804–1881), Robert Brown (1773–1858), Robert Remak (1815–1865), Rudolf Virchow (1821–1902), Oscar Hertwig (1849–1922), and others, the cell theory was established. Cells were considered as the ultimate morphological element of plants, animals, and humans [50]. From that time until recently, however, “cell” and “animalcules” observations had never clashed. Leeuwenhoek had not observed Hooke’s “cell”, nor had Hooke found “animalcules” reported by Leeuwenhoek in his “cell”. This loss of intersection for hundreds of years produced the idea of bacteria as absent from cells, and cells as absent from “animalcules”. “Cell” became the last frontier Leeuwenhoek had not touched.
Next-generation high-throughput 16S rRNA gene detection expanded the ability beyond Leeuwenhoek to observe “animalcules”. Bacteria detected in tumor cells [48, 49] reminded us of the possibility of overlaps between “cells” and “animalcules”. By means of metagenomic sequencing and in situ detection, recently we found the existence of bacteria in hepatocytes of healthy SD rats [52]. This result suggested that the bacteria in cells may be a normal life phenomenon. In this study, we further detected the existence of bacteria in parenchymal cells of various visceral organs. We performed 16S rRNA gene sequencing detection in rats (www.biomarker.com.cn, BMK200916–AC763–0101 and BMK210512–AJ541–ZX01-0101) and repeated the in situ detection more than three times. Although it is surprising, the results supported the fact that bacteria were located in parenchymal cells in rats.
Several studies have reported the positive detection of 16S rRNA genes in tissues and circulation [44, 45, 60–62], but in situ detection was less frequently performed. Moreover, these studies considered bacteria from the gut microbiota, and a comparison of the tissue bacteria with the gut microbiota was lacking. In this study, in situ detection confirmed the tissue bacteria were in fact intracellular. Interestingly, gram-negative bacteria were located in the nucleus while gram-positive bacteria were located in the cytoplasm. By comparing the visceral microbiota with their gut microbiota, we showed that the cellular microbiota in visceral organs differed from gut microbiota structurally and functionally, despite being the same species. The number of OTUs, richness, evenness, and similarity in visceral bacteria were even higher than those of the small intestine contents and feces. A high abundance of 16S rRNA genes were also detected in newborn rats and fetuses, confirming that the visceral bacteria had no relationship to the gut microbiota. This challenges the concept that the tissue bacteria came from gut microbiota.
Owing to the high throughput and high sensitivity of 16S rRNA gene sequencing, and owing to the belief that visceral organs and parenchymal cells are sterile, the tissue microbiota has caused serious concerns of contamination [63, 64]. Furthermore, the positive detection of the 16S rRNA gene does not necessarily indicate the existence of living bacteria, much less the microbiota. In this study, we performed experiments strictly according to the recommended RIDE (Report methodology, Include controls, Determine the limit of detection, and Explore the impacts of contamination in downstream analysis) checklist [48, 63] to avoid and exclude the possible contamination of bacteria and 16S rRNA gene during sampling, DNA extraction, and polymerase chain reaction (PCR), as well as the sequencing process. Detailed information and the checklist were reported in the methods [52]. Considering contamination, it is unlikely that the sampling procedure caused the visceral organs to have a higher abundance and richness of bacteria than the feces. LPS, LTA, and EUB338 probes are widely used biomarkers [65–72] for visualizing the entire bacterial population in a specimen. LPS and LTA are components anchored in the wall of bacteria, and the probe EUB 338 is complementary to a portion of the 16S rRNA gene. Antibodies (#HM6011, #HM2048; Hycult) to LPS and LTA and the probe EUB 338 have been reported in in situ histological detection by immunohistochemistry. Those results showed LPS-positive but LTA-negative reactions in various tumors [48]. In this study, we performed FISH and IF on visceral organ tissues. Compared with the negative controls, the parenchymal cells of visceral organs showed positive reactions to LPS, LTA, and the probe EUB 338. To verify the cellular location of bacteria in cells, we further performed western blotting on cytoplasm and nuclear extracts of tissues and cultured HepG2, Huh-7, Hepa1-6, and HSC-T6 (rat hepatic stellate) cells. The results showed that LPS was located in the nucleus while LTA was located in the cytoplasm (Fig. 3). These results suggested that the bacteria detected in visceral organs were intracellular inhabitants rather than from contamination.
In male adult rats, the visceral bacteria showed higher similarity and were consistent among rats compared to gut microbiota. Only very few annotated bacteria were exclusive to the brain, heart, lung, kidney, and spleen, suggesting that visceral bacteria possessed universal characteristics in the rats. However, the dominant bacteria differed across organs within an individual, and differed across individuals for a given organ, suggesting the uniqueness of visceral bacteria. These features were also shown in pregnant rats (Figures S15–S18). Owing to sampling difficulties, organs in newborn rats and fetuses could not be detected individually. However, they all showed a high abundance of 16S rRNA genes. Results showed that the visceral bacteria in newborn rats were similar to those of the milk clot. Milk bacteria have been reported recently [73–79], and thus can be a source of gut microbiota of an individual [80–82]. The visceral bacteria in the fetuses had the same abundance and alpha diversity as their mothers; however, the beta diversity analysis results differed from their mothers (Figure S14), suggesting the fetal and placental bacteria were distinct and perhaps from the initial zygote cells. Placental and fetal bacteria have also been reported recently [83–87]. According to those reports and our result of the beta diversity analysis, it can be deduced that the placenta microbiota should be part of the embryo rather than part of mother gut microbiota.
Although the results sound reasonable, it is almost inconceivable and very difficult to draw a conclusion that bacteria are located in the parenchymal cells, especially in the nucleus. For decades, cytologists, histologists and pathologists had never reported the special morphology of cellular bacteria. Were they limited by the no-intersection observation since the birth of the “cell” and “animalcules” and ignored the observed bacteria? Or perhaps the morphological features of intracellular bacteria are different from those of external bacteria? In fact, there is a large number of mystery particles in the cell that are only now are being recognized, such as the exosomes. If the “cell” had been observed by Leeuwenhoek, there may be completely different “animalcules” from those found in nature. The morphological features of cellular microbiota need to be established. The cellular bacteria provide new insight into life processes and pathological mechanisms of diseases, including the occurrence and development of tumors. The cell was the original unit of life. After the organelles, molecules, and pathways were added, we propose that the cellular microbiota is the next system to be added. Several basic questions may be arising. Where are the bacteria from? Are there actual bacteria or are these findings simply inherent structural components of the cells? How does this tiny life coexist in the cells? Why are they located in different parts of the cell, with LPS in the nucleus and LTA in the cytoplasm? What is the cellular microbiota function in genomic replication, transcription, translation, or epigenetic regulation of host cells? What functions do they undertake as part of the normal activities of the cell and in the development of tumors or occurrence of hereditary diseases? It can be speculated that the bacteria were sealed by cell membrane structure during the cell origination and were transferred to progeny cells during cell division. In addition to the cellular genome, the microbiota is another set of genetic material transferred from a cell to its new generation.
In 1858, Virchow gave the most adequate definition of cellular pathology as follows: “Each disease originates from the alterations that affect a smaller or larger number of cellular units within the living organism; every pathological disturbance, every therapeutic effect can only then ultimately have interpreted, when it is possible to tell which particular group of living cellular elements is concerned, and which kind of alterations each element of such a group has undergone. The long searched for essence of disease is the altered cell." [50] Now, it should be the altered cellular microbiota. Do the intracellular bacteria cause intracellular disease, such as degeneration, fibrosis, and tumors, while extracellular bacteria, from the external environment, destroy cells?
In conclusion, this article reported that cellular microbiota resided in parenchymal cells as inherent inhabitants in visceral organs of rats. They were cellular intrinsic inhabitants rather than translocated through gut leakage or the fetal blood barrier. Further study is needed to identify these endogenous intracellular bacteria in different organs.