Does SARS-CoV-2 have a “bacteriophage-like” mechanism of action?

It was thought that the only possible host for SARS-CoV-2 is mammalian eukaryotic cells. The Italian authors conducted a series of experiments to determine the presence of SARS-CoV-2 in the cultivated fecal microbiota of infected individuals and to examine the correlation between fecal bacteria and the virus. The findings revealed a significant increase in the RNA load of SARS-CoV-2 in bacterial cultures. A microscopic analysis revealed virus-like particles near and inside the bacteria, whereas the utilization of anti-SARS-CoV-2 nucleocapsid protein antibody for immunogold labelling revealed the presence of gold nanoparticles within the bacteria. The authors noted that these findings suggest a “bacteriophage-like” behavior of SARS-CoV-2, which, to their knowledge, has not previously been observed or described.

The SARS-CoV-2 RNA has been found in human fecal specimens, including those from people with typical COVID-19 symptoms and multiple negative nasopharyngeal SARS-CoV-2 PCR swabs.

The bacteriophage is a virus that infects bacterial cells. These viruses are largely considered inert with respect to the human host due to the absence of specific receptors for bacteriophages in eukaryotic cells. 

About the studies

The first experimental study was designed to investigate the survival of SARS-CoV-2 over time, the eventual multiplication of viral RNA in vitro, the synthesis of the SARS-CoV-2 peptides in the cultures with confirmed viral RNA presence, the consequences of antibiotic administration, and the presence of eukaryotic cells in human fecal specimens.

The study included two fecal samples, one from a person who was positive to SARS-CoV-2 and another from a healthy person (referred to as samples A and B, respectively). Sample B was inoculated with the supernatant of sample A, which was obtained through centrifugation (called sample B_(A+)). All specimens were incubated for 30 days under the same conditions, and the viral RNA was measured in each specimen on days 1, 2, 3, 7, 14, 21, and 30, following the date of inoculation (day 0).

On day 21, 18 antibiotics were added to 18 aliquots derived from sample B_(A+): metronidazole, clindamycin, lincomycin, piperacillin+tazobactam, vancomycin, amoxicillin, ampicillin, cefixime, ceftriaxone, meropenem, rifaximin, azithromycin, erythromycin, gentamicin, ciprofloxacin, colistin, levofloxacin, and teicoplanin. The RNA load of SARS-CoV-2 was measured before and three days after the administration of antibiotics.

The authors observed a significant increase in the viral RNA load in sample B_(A+) and a slight increase in the viral RNA load in sample A over time. As expected, the viral RNA load in sample B was constantly negative. Extra-corporal multiplication of SARS-CoV-2 RNA has been confirmed by these findings.

The entire experiment was repeated three times with the same specimens to ensure the reproducibility of results. The initial experiment’s findings were confirmed, with the viral RNA load increasing over time in samples A and B_(A+), whereas the viral RNA load in sample B remained negative.

Also, three additional pairs of fecal specimens from different infected individuals (referred to as A1, A2 and A3) and healthy individuals (referred to as B1, B2 and B3) in all combinations of specimens were treated with the same experimental procedure. Despite certain differences, the results confirmed that the viral RNA load increased over time in samples of type A and type B_(A+). The RNA load of SARS-CoV-2 was particularly high in the A2×B2 combination.

The experiment revealed that samples A and B_(A+) contained some bacterial genera that were particularly abundant and metabolically active.

Additionally, the results showed that the RNA load of SARS-CoV-2 in aliquots derived from sample B_(A+) changed depending on the antibiotic that was added to them. A reduction of viral RNA load to an undetectable level was observed in four aliquots treated with metronidazole, vancomycin, amoxicillin and azithromycin. The viral RNA load decreased from 20% to 85% in aliquots treated with piperallicin+tazobactam, ampicillin, cefixime, ceftriaxone, meropenem, gentamicin, ciprofloxacin and teicoplanin . Cefixime reduced the viral RNA load by 85%, ciprofloxacin by 61% and teicoplanin by 56%. In aliquots treated with clindamycin, lincomycin, rifaximin, erythromycin, colistin and levofloxacin, viral RNA load did not change.

During their subsequent analysis, researchers used a transmission electron microscope and a scanning electron microscope to examine the presence of eukaryotic cells in samples A, B, and B_(A+) collected at various times. Only bacterial cells were found in more than 30 different preparations, without any structure that resembled cells with nuclei. The images of samples A and B_(A+) revealed the presence of virus-like particles that interact with bacterial cells.

The researchers emphasized that they repeated the experiment three times with the same samples, and replicated the experiment using a 3×3 design with specimens from different individuals. In all experiments, the results showed a similar pattern, with the increase in RNA load of SARS-CoV-2 in samples A and B_(A+). According to the authors, these findings suggest that the SARS-CoV-2 genome is capable of replicating outside the human body. This means that SARS-CoV-2 might have a ‘bacteriophage-like’ mechanism of action, so, it is important to find out which bacterial species are targets of SARS-CoV-2.

This article was published in F1000Research.

Journal Reference

Petrillo M. t al. Increase of SARS-CoV-2 RNA load in faecal samples prompts for rethinking of SARS-CoV-2 biology and COVID-19 epidemiology. F1000Research 2021, 10:370. (Open Access) https://doi.org/10.12688/f1000research.52540.3

Recently, the Italian research group has analyzed cultures of bacteria from the human microbiome and SARS-CoV-2 by electron and fluorescence microscopy. They also conducted the nitrogen isotope 15N assay in bacterial culture medium, accompanied by proteomic analysis of SARS-CoV-2.

A microscopic analysis of the interaction between SARS-CoV-2 and the human microbiome revealed virus-like particles within and near the bacteria. Also, the utilization of anti-SARS-CoV-2 nucleocapsid protein antibody for immunogold labelling revealed the presence of gold nanoparticles within the bacteria. The initial areas of cell wall lysis and fully lysed bacteria were observed as well. The authors stated that the visualization of gold nanoparticles in bacteria provides conclusive evidence of the viral presence within the bacteria. The labeling of SARS-CoV-2 proteins with nitrogen (15N) isotopes demonstrated that bacteria are capable of replicating, transcribing, and translating viral RNA. Thus, the proteins of this virus can be produced by bacteria.

This article was published in Vaccines.

Journal Reference

Brogna, C, et al. Could SARS-CoV-2 Have Bacteriophage Behavior or Induce the Activity of Other Bacteriophages? Vaccines 2022, 10, 708. (Open Access)  https://doi.org/10.3390/vaccines10050708