Carlo Brogna 1&*, Barbara Brogna 2, Domenico Rocco Bisaccia 1, Francesco Lauritano 1, Marino Giuliano 3, Luigi Montano 4, Simone Cristoni 5, Marina Prisco 6, and Marina Piscopo 6&*.
1. Department of research, Craniomed group facility Srl., Montemiletto (Av), Italy; dir.brogna@craniomed.it (C.B.); roccobisa@gmail.com (R.D.B.); francescodrlauritano@gmail.com (F.L.)
2. Department of Radiology, Moscati Hospital, Contrada Amoretta, 83100 Avellino, Italy; brognabarbara1@gmail.com (B.B.)
3. Marsanconsulting Srl. Public Health Company; Via dei Fiorentini, 80133 – Napoli; Italy; marino@marsanconsulting.it (M.G.)
4. Andrology Unit and Service of LifeStyle Medicine in Uro-Andrology, Local Health Authority (ASL) Salerno; luigimontano@gmail.com (L.M.)
5. ISB – Ion Source & Biotechnologies Srl- Bresso Milano; simone.cristoni@gmail.com (S.C.)
6. Department of Biology, University of Naples Federico II, 80126 Napoli, Italy; marina.prisco@unina.it (M.P.); marina.piscopo@unina.it (M.P.)
* Correspondence and equal contributor: marina.piscopo@unina.it; dir.brogna@craniomed.it
Abstract: SARS-CoV-2 has become one of the most studied viruses of the last century. It was assumed that the only possible host for these types of viruses was mammalian eukaryotic cells. Our recent studies show that microorganisms in the human gastrointestinal tract affect the severity of COVID-19 and demonstrate for the first time that the virus replicates in gut bacteria. To complete this investigation, cultures of bacteria from the human microbiome and SARS-CoV-2 were analyzed by electron and fluorescence microscopy, in this work. The images presented in this paper, in association with the Nitrogen (15N) isotope-labeled culture medium experiment, clearly show that SARS-CoV-2 infects bacteria in the gut microbiota, indicating that SARS-CoV-2 acts as a bacteriophage. Our results add new knowledge to the understanding of the mechanisms of SARS-CoV-2 infection and fill gaps in the study of the interactions between SARS-CoV-2 and non-mammalian cells. These conclusions could suggest specific new pharmacological solutions to support the vaccination campaign.
Keywords: SARS-CoV-2; human microbiota; Sabin vaccine; electron microscope; mucosal immunity; bacteriophage
The curves of replication and viral load increase of SARS-CoV-2 in bacterial cultures are
shown in Figure 1, panels A and B, with gentle permission of Dr. Petrillo [7]. In particular,
three couples of faecal samples from different “infected donors” (i.e. sources of A) and
“healthy recipients” (i.e. sources of B) have been recruited, and subject to the same
experimental procedure described in materials and methods. All combinations of “infected
donors” sources (A1, A2 and A3) and “healthy recipients” donors (B1, B2 and B3) were
subject to the same experimental procedure. Although with certain differences, the observed
trends were similar (Figure 1A), confirming the increase over time of SARS-CoV-2 RNA load
in samples of type A and in samples of type B(A+). Lastly, for each “recipient”, SARS-CoV-
2 RNA load was measured (Figure 1B). The results report virus replication in bacterial
cultures suggesting that this virus behaves as a bacteriophage.
Figure 1. Panel A-B: viral increase of SARS-CoV-2 in bacterial cultures. These images are reused and correspond to figure 3 of Petrillo et al. 2021 (doi: 10.12688/f1000research.52540.1.) obtained with gentle permission of Dr. Petrillo et al. 2021[7]. Results of experiments combining samples from three “infected donor” sources (A1, A2 and A3) and from three “healthy recipient” sources (B1, B2 and B3). A) The graphs report results of nine combinations. To normalize the measurements, all values at day 0 were used as the denominator (at day 0 all values = 1), i.e. for each sample, at day X, the ratio between Luminex Count At DayX/Luminex CountAt Day 0 was calculated. Each bar represents the SARS-CoV-2 RNA load ratio. B) Each line represents the average of the SARS-CoV-2 RNA load ratio of samples B1 (green line), B2 (yellow line), and B3 (azure line) infected each one with three different A donor sources. To normalize the measurements, all values at day 0 were normalised as described in panel A). Panels C-L show the immunofluorescence microscope images (Zeiss Axioplan 2, Axiocam 305 color, magnification 100X) on bacterial cultures (performed at 30 days) from the patients positive to SARS-CoV-2 with anti-gram-positive bacteria (green signal, C) and anti-SARS-CoV-2 nucleocapsid protein (red signal, D) antibodies. E: merge of C and D images, particles positive to both antibodies were indicated by white arrows. F-I: computerized magnification of particles numbered in C-E. Panel L: negative control by omitting primary antibodies. Panel M is the merge of the negative control of a healthy fecal bacterial culture, in which the anti-bodies versus nucleocapsid protein didn’t give signal and gram+ bacteria are present.
See text for more details.
Figure 2. TEM images of bacterial cultures from SARS-CoV-2 positive patients. A-C shows circular structures (blue arrows) inside bacteria cells (I, II, III, IV). D-G: immunogold labelling tecnique on the same samples with anti-nucleocapsid protein of SARS-CoV-2 antibody (gold arrows); the gold particles are inside the bacteria. Red
arrows show lysis of the bacteria membrane. Specifically, image G shows bacteria wall membrane lysate (red arrow) and the antibody binding to SARS-CoV-2 N protein in the cell (gold arrows). H: negative control.
See text for more details.
An interactive metagenomic visualization using the tool Krona [15] is shown in Figure 3 A-B. The multilayer pie chart represents the degrees of the bacterial world. The division is from inside to outside the circle. Colors towards red, have a low level of confidence while colors towards green have a high level of confidence. The initial and final samples, after 30 days, of the bacterial cultures are compared. The genera of bacteria such as Dorea, Fusicatenibacter, Klebsiella, Streptococcus decreased while other genera of bacteria such as Campylobacter, Prevotella, Staphylococcus, Bacteroides, and Cytobacter increased. In Table 2 (supplementary material), we provide a list of other bacteriophages present after 30 days of bacterial culture. These bacteriophages differ in size, shape, and most importantly are characterized by the presence of a tail, which is absent in the viral particles we present in TEM and immunogold images. Therefore, these data give insight into which genera of bacteria can undergo SARS-CoV-2 lysis and in particular make it possible to exclude other bacteriophages characterized by accessory microscopic structures that the virions shown do not possess.
After 30 days of bacterial growth in liquid medium, nitrogen (15N) isotope-labeled medium (Sigma Aldrich) was added. After an additional 7 days, proteomic analysis of SARS-CoV-2 was performed. Modified peptides of the SARS-CoV-2 nucleocapsid protein were detected by extracting the 13C n15N species as specified in Materials and methods The spectra obtained by Tandem mass spectrometry (MS/MS) [16] and acquired between 200 and 1800 m/z (mass/charge), show fragmentation of modified peptide of Nucleocapsid SARS-CoV-2 protein, containing 13C and 15N isotopes. The sequences are shown in figure 4 A-C. The nitrogen isotope (15N) was detected in nucleocapsid peptide of SARS-CoV-2 replicated in bacterial cultures by monitoring the molecular mass shift. The MS/MS spectra shows that the 15N species is located on aminoacid residue of peptides. Nitrogen (15N) isotope labeling in SARS-CoV-2 proteins demonstrates that bacteria can replicate, transcribe, and translate viral RNA and thus that bacteria produce the proteins of this virus.
Figure 3. Panel A: represents the phylum of bacteria present in the culture before the addition of SARSCoV-2 while panel B represents the change in bacterial population after 30 days of the same cultures infected with SARS-CoV-2 .
For more detail see supplementary materials.
Figure 4. Panels A-C: shows the MS/MS spectra of the nucleocapsid protein of SARS-CoV-2 containing
the nitrogen isotope. Panel A shows peptide seq: QQSMSSADSTQA;
ID:|A0A8B1JYE4|A0A8B1JYE4_SARS2; Mods: Q408 + 1 (13C) +2 (15N), Q409 + 1 (13C) +2 (15N) . Panel
B shows peptide seq: NPANNAAIVLQL ; ID: A0A7M1YDW6|A0A7M1YDW6_SARS2;; Mods: Q160+
1 (13C) +2 (15N). Panel C shows peptide seq: QQQQCQTVTKK; ID:|A0A8B1XSI6|A0A8XSI6_SARS2;
Mods: Q239+ 1 (13C) +2 (15N), Q239+Ammonia-loss, Q240+ 1 (13C) +2 (15N), Q241+ 1 (13C) +2 (15N),
Q242+ 1 (13C) +2 (15N), Q244+ 1 (13C) +2 (15N).
In vitro bacterial cultures were obtained from fecal (stool) samples from subjects both
SARS-CoV-2 positive and negative (for control cases) for viral infection. Informed consents
from patients were obtained according to local legislation. Replication of virus in human
bacteria by statistical analysis, Luminex molecular tests, and further controls by mass
spectrometry technique (SACI technique) are reported in Petrillo et al., 2021 [7]. Immunogold
was performed with the bacteria culture pellet at day 30 after centrifugation of the aliquots.
Supernatants were used from the same samples to acquire immunofluorescent images using a
fluorescence microscope (100X magnification, Zeiss Axioplan 2, Axiocam 305 color
supplementary materials). Each step was repeated three times.
Another pellet of 0,5 mL of the same bacterial cultures was fixed by resuspending with
0.05% glutaraldehyde and 4% paraformaldehyde in 0.1 M PBS buffer, and then centrifugate
to 10000 rpm for ten minutes. Thereafter, the bacteria pellets were post-fixed in 1% OsO4, and
processed according to the standard procedures, by dehydration in ethanol followed by
infiltration and embedding in Epon resin and polymerization at 60°C for 48 hours. Ultrathin
(60-nm) sections were cut from the sample using a Reichert-Jung ultramicrotome and
collected onto Formvar/Carbon coated nickel grids. After etching with 3% sodium meta
periodate and blocking with 3% BSA in PBS buffer, the samples were incubated in a primary
rabbit monoclonal [EPR24334-118] to SARS-CoV-2 nucleocapsid protein antibody (Abcam
ab 271180) 1:1000 in 1% BSA in PBS buffer overnight at 4°C and then in anti-rabbit 10 nm
gold-conjugated secondary antibody (Aurion) 1:20 in PBS buffer for 2 hours at room
temperature.
Negative control was obtained by omitting the primary antibody and using only the
secondary gold-conjugate antibody. The control of immunogenicity of antibodies primary
versus nucleocapsid protein of SARS-CoV-2 was assumed from Shang C. et al. [32].
Grids were counterstained with UranyLess EM stain and Lead Citrate 3% (Electron
Microscopy Science) and images were acquired under a transmission electron microscope
(FEI Tecnai G2 S-TWIN) equipped with a bottom-mounted FEI Eagle4k digital camera.
Bacteria pellets (0,5 mL) were fixed by resuspending with a mixture of 0.05% glutaraldehyde
of 4% paraformaldehyde in 0.2M HEPES buffer. Then bacteria pellet, obtained by
centriguation ( 10000 rpm for ten minutes), were resuspended, washed with PBS, and
incubated first in blocking solution (50mM NH4Cl, 0.1% saponin, 1%BSA in PBS) and then
in primary antibody against Nucleocapsid SARS-CoV-2 (N) protein (Abcam, #ab273167).
Then the cells were washed with PBS and incubated with NANOGOLD conjugated anti-rabbit
Fab fragments (Nanoprobes, #2004). After washes in PBS and distilled water bacterial pellets
were kept in the gold-enhancement mixture (Nanoprobes). Immunolabelled specimens were
post-fixed in OsO4 and uranyl acetate and embedded in EPON. Thin sections were cut from
embedded specimens using Leica EM UC7 (Leica Mycrosystems). Electron microscopy
images were acquired from thin sections under an electron microscope (Tecnai G2 Spirit
BioTwin; FEI) equipped with a VELETTA CCD digital camera (Soft Imaging Systems
GmbH).
cDNA encoding green fluorescent protein (GFP)-tagged N-protein of SARS-CoV-2 was
generously provided by Dr. M.A. De Matteis (TIGEM, Pozzuoli, Italy) and transfected into
HeLa cells using TransIT-LT1 reagent (Mirus Bio LLC) according to the manufacture
instruction. Transfected cells were fixed in 4% paraformaldehyde, washed with PBS and
incubated in blocking solution (50mM NH4Cl, 0.1% saponin, 1%BSA in PBS). Then the cells
were labelled using primary antibody against N protein (Abcam, #ab273167) and secondary
anti-rabbit IgG conjugated with Alexa Fluor 568 (Thermo Fisher; # A-11011). After labelling
the samples were examined under a LSM700 confocal microscope (Zeiss). Efficiency of
antibody against N-protein was confirmed based on the presence of Alexa Fluor 568 signal in
the cells expressing GFP-tagged N-protein.
Aliquots of the supernatants of the samples were obtained and the slices were fixed with
4% PFA for 5 minutes. After permeabilization with Triton 0.3% in PBS for 10 minutes, the
slices were rinsed in PBS and blocked with BSA 1% and saponin 0.05% for 30 minutes. The
slices were incubated with the primary antibodies for 2 hours at room temperature and then
with secondary antibodies for 1 hour at room temperature. Immunofluorescence were
performed according to the manufacturer’s protocol using a primary antibody against the virus
nucleocapsid protein („Sars Nucleocapsid Protein Antibody [Rabbit Polyclonal] – 500μg 200-
401-A50 Rockland“), the „Goat anti-RabbitIgG (H+L) Cross-Adsorbed Secondary Antibody,
Cyanine3 #A10520″ as a secondary antibody, and a primary antibody against gram-positive
bacteria („Gram-Positive Bacteria Ab (BDI380), GTX42630 Gene Tex“) and „Goat anti-
Mouse IgG (H+L), Super-clonal™ Recombinant Secondary Antibody, AlexaFluor 488″ as a
secondary antibody“. The negative control of the bacterial cultures was performed without
primary antibodies versus gram-positive bacteria and without primary antibodies versus
nucleocapsid SARS-CoV-2 proteins. The control of immunogenicity of primary antibodies
versus bacteria Gram-positive was performed on the culture negative at Luminex molecular
test to SARS-CoV-2. It was also assume the control of immunogenicity of antibodies versus
Gram-positive bacteria from Kohda et al. and Kameli et al. [33-34]. The control of
immunogenicity of primary antibodies versus nucleocapsid protein of SARS-CoV-2 was
assume from Zhao et al. [35].
One bacterial liquid culture was used to add, at the end of 30 days, 0,20 gr. of a nitrogen
isotope (15N) labeled medium ( Sigma Aldrich). After another 7 days, a SARS-CoV-2
proteomic analysis was performed, on aliquots (0,2 mL) of the same samples by SACI
technology [16]. Modified peptides of nucleocapsid SARS-CoV-2 protein were detected by
extracting the species 13C n15N ( where n>1). Ion proton rearrangement reaction occurring in
Collisional Induced Dissociation (CID) conditions were considered in the database search data
elaboration [36].The ion were isolated using 5 m/z units and the fragmentation energy was
35% of its maximum value (5V peak to peak).
DNA from fecal samples, at day 0 (B0) was extracted with E.Z.N.A.® Stool DNA Kit (Omega
Bio-Tech, GA, USA). Two types of kit for the nucleic acid extractions from samples after
30 days (B1) of culture were used: MasterPure Complete DNA and RNA purification kit
(Lucigen, WI, USA) and PureLink Viral RNA/DNA kit (Thermo Fisher Scientific, MA,
USA). All extractions were performed following the manufacturers‘ recommendations. The
Ovation® Ultralow V2 DNA-Seq Library Preparation kit (NUGEN, San Carlos, CA) was
used for library preparation, following the manufacturer’s instructions. Both input and final
libraries were quantified with the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA), and
quality was tested by Agilent 2100 Bioanalyzer High Sensitivity DNA assay (Agilent
Technologies, Santa Clara, CA). Libraries were then prepared for sequencing and sequenced
on NovaSeq 6000 in 150-bp paired-end mode. Bioinformatic reconstruction was calculated
on the fasta file of bacterial sequences on UniProt. Result are presented in Table 1, in
supplemetary material. In addition to exlude other bacteriophage viruses particle the
bioinformatic reconstruction was calculated also on the fasta file of bacteriophage sequences
on UniProt (Table 2, in suplementary materials).
The control of prokaryotic cells as a reservoir source for some viruses represents an
element that should be the basis of any diagnostic pathway regardless of whether one is
convinced that it can occur or not. Our results obtained by Immuno-EM, embedded
immunogold, and fluorescence imaging highlight a close connection between the coronavirus
SARS-CoV-2 and bacteria of the intestinal microflora. SARS-CoV-2 has two mechanisms of
action; it infects both the eukaryotic cell, as reported in the current literature, but it also infects
the human bacterial flora (two bacteria are already identified, data in preparation) and Sabin’s
solution, the attenuated vaccine for oral administration, could be a solution that could
complement the vaccination campaign to exit definitively from this pandemic. Our results
confirm the role of bacterial co-factors in the multiplication of COVID 19 coronavirus during
this new epidemic confirmed by the efficacy of some previously described antibiotics [7,8]
on the early phase of viral infection. Whether it also plays a role in long-term disease remains
to be determined. In this case, we could consider the virus as a bactériophage with a lytic
phase and a lysogenic phase. Furthermore, the presence of the nitrogen isotope 15N in viral
proteins after infection of bacteria with SARS-CoV-2 could represent a new approach to study
the interaction of RNA viruses with human microflora.
Author Contributions: “Conceptualization, C.B. and M.P. (Marina Piscopo); methodology, C.B. D.B. L.F.; software, C.B, B.B., L.F.; validation, M.P., S.C.; formal analysis, S.C. and M.P.( Marina Prisco); investigation, C.B:, S.C..; resources, C.B. and G.M.; data curation, M.P.( Marina Piscopo) and C.B..; writing—original draft preparation, C.B. and M.P..; writing—review and editing, M.P..; visualization. M.P., and C.B..; supervision, C.B., and M.P.; project administration, C.B..; funding acquisition, G.M., C.B. All authors have read and agreed to the published version of the manuscript.”
Funding: Please add: “This research received no external funding”
Institutional Review Board Statement: the study did not require ethical approval.
Informed Consent Statement: “Informed consent was obtained from all subjects involved in the study.”
Data Availability Statement: “Not applicable” here.
Acknowledgments: I thank Dr. M. Petrillo, Dr. G. Ciammetti, Prof. O. Piazza (University of Medicine in Salerno). I also thank the Biogem Institute of Ariano Irpino (Av) for fluorescence microscope images. We would like to acknowledge TIGEM Advanced Microscopy and Imaging Core for the help with preparation of specimens for fluorescent and electron microscopy. We also are grateful to the Electron microscopy facility of the department of chemical sciences- University of Study Federico II Naples (UniNa). I also am grateful to Marsan consulting and Dr. Marino Giuliano for their full support.
Conflicts of Interest: “The authors declare no conflict of interest.”
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3. Interview mit Dr. Carlo Brogna Arzt und Corona Virus Forscher
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5. MIKE YEADON, EX-VIZEPRÄSIDENT VON PFIZER PACKT AUS: „WIR STEHEN AN DEN PFORTEN DER HÖLLE.“
6. Erstmaliger Nachweis der Impf-Spikeprotein bei einernach der Impfung gegen Covid 19 verstorbenen Patienten
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