Running after ghosts: are dead bacteria the dark matter of the human gut microbiota?

The human gut microbiota has been explored by a wide range of culture-dependent and culture-independent methods, revealing that many microbes remain uncharacterized and uncultured. In this work, we aimed to confirm the hypothesis that some of the species present in the human gut microbiota remain uncultured not because of culture limitations, but because all members of such species are dead before reaching the end of the gastro-intestinal tract.We evaluate this phenomenon by studying the microbial viability and culturability of the human gut microbiota from the fresh fecal materials of eight healthy adults. For the first time, we applied fluorescence-activated cell sorting (FACS) combined with 16S metagenomics analysis and microbial culturomics.We identified a total of 1,020 bacterial OTUs and 495 bacterial isolates through metagenomics and culturomics, respectively. Among the FACS metagenomics results, only 735 bacterial OTUs were alive, comprising on average 42% of known species and 87% of relative abundance per individual. The remaining uncultured bacteria were rare, dead, or injured.Our strategy allowed us to shed light on the dark matter of the human gut microbiota and revealed that both metagenomics and culturomics approaches are needed for greater insight into the diversity and richness of bacteria in the human gut microbiota. Further work on culture is needed to enhance the repertoire of cultured gut bacteria by targeting low abundance bacteria and optimizing anaerobic sample conditioning and processing to preserve the viability of bacteria.

High-speed large-scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled to high-content screening.

OBJECTIVES: A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the current coronavirus disease 2019 global pandemic. Only a few laboratories routinely isolate the virus, which is because the current co-culture strategy is highly time-consuming and requires a biosafety level 3 laboratory. This work aimed to develop a new high-throughput isolation strategy using novel technologies for rapid and automated isolation of SARS-CoV-2. METHODS: We used an automated microscope based on high-content screening (HCS), and we applied specific image analysis algorithms targeting cytopathic effects of SARS-CoV-2 on Vero E6 cells. A randomized panel of 104 samples, including 72 that tested positive by RT-PCR and 32 that tested negative, were processed with our HCS strategy and were compared with the classical isolation procedure. RESULTS: The isolation rate was 43% (31/72) with both strategies on RT-PCR-positive samples and was correlated with the initial RNA viral load in the samples, in which we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with the HCS strategy, where 80% (25/31) of the positive samples were recovered by the third day of co-culture, compared with only 26% (8/30) with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive samples (31 with HCS versus 27 with classic strategy) after 1 week of co-culture. CONCLUSIONS: This system allows the rapid and automated screening of clinical samples with minimal operator workload, which reduces the risk of contamination and paves the way for future applications in clinical microbiology, such as large-scale drug susceptibility testing.

Rapid Scanning Electron Microscopy Detection and Sequencing of Severe Acute Respiratory Syndrome Coronavirus 2 and Other Respiratory Viruses.

There is an urgent need for accurate and rapid testing methods to quickly identify infected patients as well as asymptomatic carriers, in order to prevent the spread of emerging viruses. Here, we developed a rapid testing strategy by scanning electron microscopy capable of detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses directly from patients. We evaluated our results by comparing them to real-time reverse transcription-polymerase chain reaction (RT-PCR) and metagenomic sequencing results. We correlated the presence of the SARS-CoV-2 to the viral load, where samples with Ct values lower than 18 were all detected by scanning electron microscopy (SEM). The sensitivity deacresed progressively with higher Ct values. In addition, we found a correlation with metagenomic sequencing, where all samples detected by SEM were sequenced and viral sequences were easily recovered. Following this study, SEM proved its efficiency as a frontline method for directly detecting previously unknown microorganisms that cannot be targeted by molecular methods and can cause potential outbreaks.

Advantages and Limits of Metagenomic Assembly and Binning of a Giant Virus.

Giant viruses have large genomes, often within the size range of cellular organisms. This distinguishes them from most other viruses and demands additional effort for the successful recovery of their genomes from environmental sequence data. Here, we tested the performance of genome-resolved metagenomics on a recently isolated giant virus, Fadolivirus, by spiking it into an environmental sample from which two other giant viruses were isolated. At high spike-in levels, metagenome assembly and binning led to the successful genomic recovery of Fadolivirus from the sample. A complementary survey of the major capsid protein indicated the presence of other giant viruses in the sample matrix but did not detect the two isolated from this sample. Our results indicate that genome-resolved metagenomics is a valid approach for the recovery of near-complete giant virus genomes given that sufficient clonal particles are present. However, our data also underline that a vast majority of giant viruses remain currently undetected, even in an era of terabase-scale metagenomics.IMPORTANCE The discovery of large and giant nucleocytoplasmic large DNA viruses (NCLDV) with genomes in the megabase range and equipped with a wide variety of features typically associated with cellular organisms was one of the most unexpected, intriguing, and spectacular breakthroughs in virology. Recent studies suggest that these viruses are highly abundant in the oceans, freshwater, and soil, impact the biology and ecology of their eukaryotic hosts, and ultimately affect global nutrient cycles. Genome-resolved metagenomics is becoming an increasingly popular tool to assess the diversity and coding potential of giant viruses, but this approach is currently lacking validation.

Coculture at the crossroads of the new microbiology techniques for the isolation of strict intracellular bacteria.

Coculture played a major role in clinical microbiology by elucidating strict intracellular bacteria era. Since some of these bacteria are human pathogens, in-depth studies at the strain level are necessary to better understand their pathologies and treatment. Over the last decades, culture-independent tools have taken over the diagnostic procedure at the expense of coculture. These tools, although proven to be rapid and efficient, cannot overcome the need to culture the bacteria, as strain isolation remains a key factor to understanding its epidemiology, virulence and antibiotic susceptibility testing. Moreover, strain availability allows the development of molecular and serological tools, and remains crucial for taxonomy. This review revisits the current status of culture, its advantages, drawbacks and future needs.

High-Content Screening, a Reliable System for Coxiella burnetii Isolation from Clinical Samples.

Q fever, caused by Coxiella burnetii, is a worldwide zoonotic disease that may cause severe forms in humans and requires a specific and prolonged antibiotic treatment. Although current serological and molecular detection tools allow a reliable diagnosis of the disease, culture of C. burnetii strains is mandatory to assess their susceptibility to antibiotics and sequence their genome in order to optimize patient management and epidemiological studies. However, cultivating this fastidious microorganism is difficult and restricted to reference centers, as it requires biosafety level 3 laboratories and relies on cell culture performed by experienced technicians. In addition, the culture yield is low, which results in a small number of isolates being available. In this work, we developed a novel high-content screening (HCS) isolation strategy based on optimized high-throughput cell culture and automated microscopic detection of infected cells with specifically designed algorithms targeting cytopathic effects. This method was more efficient than the shell vial assay, at the level of time dependency, when applied to both frozen specimens (7 isolates recovered by HCS only, sensitivity 91% versus 78% for shell vial) and fresh samples (1 additional isolate using HCS, sensitivity 7% versus 5% for shell vial), for which most strains were recovered more rapidly with the new technique. In addition, detecting positive cultures by an automated microscope reduced the need for expertise and saved 24% of technician working time. Application of HCS to antibiotic susceptibility testing of 12 strains demonstrated that it was as efficient as the standard procedure that combines shell vial culture and quantitative PCR.

High-throughput isolation of giant viruses using high-content screening.

The race to discover and isolate giant viruses began 15 years ago. Metagenomics is counterbalancing coculture, with the detection of giant virus genomes becoming faster as sequencing technologies develop. Since the discovery of giant viruses, many efforts have been made to improve methods for coculturing amebas and giant viruses, which remains the key engine of isolation of these microorganisms. However, these techniques still lack the proper tools for high-speed detection. In this paper, we present advances in the isolation of giant viruses. A new strategy was developed using a high-throughput microscope for real-time monitoring of cocultures using optimized algorithms targeting infected amebas. After validating the strategy, we adapted a new tabletop scanning electron microscope for high-speed identification of giant viruses directly from culture. The speed and isolation rate of this strategy has raised the coculture to almost the same level as sequencing techniques in terms of detection speed and sensitivity.

Discovery and Further Studies on Giant Viruses at the IHU Mediterranee Infection That Modified the Perception of the Virosphere.

The history of giant viruses began in 2003 with the identification of Acanthamoeba polyphaga mimivirus. Since then, giant viruses of amoeba enlightened an unknown part of the viral world, and every discovery and characterization of a new giant virus modifies our perception of the virosphere. This notably includes their exceptional virion sizes from 200 nm to 2 µm and their genomic complexity with length, number of genes, and functions such as translational components never seen before. Even more surprising, Mimivirus possesses a unique mobilome composed of virophages, transpovirons, and a defense system against virophages named Mimivirus virophage resistance element (MIMIVIRE). From the discovery and isolation of new giant viruses to their possible roles in humans, this review shows the active contribution of the University Hospital Institute (IHU) Mediterranee Infection to the growing knowledge of the giant viruses' field.