Molecular Studies Neglect Apparently Gram-Negative Populations in the Human Gut Microbiota

Abstract

dStudying the relationships between gut microbiota, human health, and diseases is a major challenge that generates contradictory results. Most studies draw conclusions about the gut repertoire using a single biased metagenomics approach. We analyzed 16 different stool samples collected from healthy subjects who were from different areas, had metabolic disorders, were immunocompromised, or were treated with antibiotics at the time of the stool collection. The analyses performed included Gram staining, flow cytometry, transmission electron microscopy (TEM), quantitative real-time PCR (qPCR) of the Bacteroidetes and Firmicutes phyla, and pyrosequencing of the 16S rRNA gene amplicons targeting the V6 region. We quantified 10 10 prokaryotes per gram of feces, which is less than was previously described. The Mann-Whitney test revealed that Gram-negative proportions of the prokaryotes obtained by Gram staining, TEM, and pyrosequencing differed according to the analysis used, with Gram-negative prokaryotes yielding median percentages of 70.6%, 31.0%, and 16.4%, respectively. A comparison of TEM and pyrosequencing analyses highlighted a difference of 14.6% in the identification of Gram-negative prokaryotes, and a Spearman test showed a tendency toward correlation, albeit not significant, in the Gram-negative/Gram-positive prokaryote ratio ( ‫؍‬ 0.3282, P ‫؍‬ 0.2146). In contrast, when comparing the qPCR and pyrosequencing results, a significant correlation was found for the Bacteroidetes/Firmicutes ratio ( ‫؍‬ 0.6057, P ‫؍‬ 0.0130). Our study showed that the entire diversity of the human gut microbiota remains unknown because different techniques generate extremely different results. We found that to assess the overall composition of bacterial communities, multiple techniques must be combined. The biases that exist for each technique may be useful in exploring the major discrepancies in molecular studies.

Clostridium butyricum : from beneficial to a new emerging pathogen

“…Species level was defined with a minimum sequence identity of 98.7% as the best BLAST hit [17]. Second, it has recently been shown that molecular studies overlooked Gram-negative populations as well as minority species [60]. Enteropathogens that can be present with a subdominant status (<10 5 bacteria/mL) may therefore be neglected, adding to the difficulty in achieving clear, reproducible detection [15].…”

The Rebirth of Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota

“…In a previous study, of the 48 species, ϳ50% were detected only by cloning, 20% were identified by both techniques, and 30% were identified only by culture (192). A gap between molecular studies including pyrosequencing of the 16S rRNA gene and direct observation (16) was highlighted by Hugon et al, who emphasized that pyrosequencing neglected nearly 15% of apparently Gram-negative prokaryotes (16). The team of Jeffrey Gordon also demonstrated that culturing of a large repertoire of an individual's gut microbiota by using straightforward anaerobic culturing conditions was possible (193).…”

“…Nevertheless, electron microscopy has been used more in bacterial research to observe microorganisms for the first time, to study the structure and function of cells (25), or by environmental microbiologists (26)(27)(28). Bacterial morphologies (29) and cell wall structures can be easily observed, and Gramnegative and Gram-positive types of bacteria can be distinguished (16). Over the past decade, cryo-electron microscopy techniques, which can be divided into cryo-electron tomography (to visualize cell structures at the protein level) (29), single-particle cryo-electron microscopy (the most commonly used) (30), and electron crystallography, are techniques based on cryofixation that emerged from sample preparation steps, which can affect specimens (30).…”

“…In addition, Gram staining is not constantly a robust method because the genera Bacillus, Gemella, and Listeria (12, 13) and certain Gram-positive anaerobes (14) can display aberrant Gram staining and appear Gram variable or Gram negative (15). In fact, in a recent study of the human gut microbiota, Hugon et al revealed that electron microscopy, which is considered a reference method for studying the bacterial cell wall, allowed the identification of an average of 2-fold more Gram-positive prokaryotes than did Gram staining (16). Ziehl-Neelsen staining detects acid-alcohol-resistant bacteria, such as Mycobacterium spp.…”

Oral and Systemic Effects of Xylitol Consumption

“…Due to the variety of factors, this hugely popular concept of gut microbiome research has several flaws. Some studies revealed that microbiome alteration due to diet is largely dependent on genetics [O'Connor et al, 2014] and the analysis technique [Hugon et al, 2013]. Thus, the true diversity of the human gut microbiome remains unknown, and using fecal analyses to estimate the gut microbiome can be a major source of bias in understanding the causal role of the microbiome in human health [Janket et al, 2018].…”