The first report on detecting SARS-CoV-2 inside bacteria of the human gut microbiome: A case series on asymptomatic family members and a child with COVID-19 A case series on family members and a

Many studies report the importance of using feces as source sample for detecting SARS-CoV-2 in patients with COVID-19 symptoms but who are negative to oropharyngeal/ nasopharyngeal tests. Here, we report the case of an asymptomatic child whose family members had negative results with the rapid antigen nasopharyngeal swab tests. The 21-month-old child presented with fever, diarrhea, bilateral conjunctivitis, and conspicuous lacrimation. In this study, analysis for the presence of SARS-CoV-2 clinical gastrointestinal and neurological symptoms, combined with efficient highly sensitive molecular testing on feces, represent an efficient approach for detecting SARS-CoV-2, and for providing the correct therapy in challenging COVID-19 cases, like the one here reported. Brogna al. The first report on detecting SARS-CoV-2 inside human fecal-oral bacteria: A case series on asymptomatic family members and a child with COVID-19 This manuscript by Brogna C et al. reports the first observation of SARS-CoV-2 “inside human fecal-oral bacteria” suggesting that SARS-CoV-2 can infect bacteria like a bacteriophage. This study is inspired by a symptomatic 21-month-old child whose nasopharyngeal sample tested negative on a rapid antigen test, but feces tested positive for SARS-CoV-2 viral RNA on a Luminex assay. This led the authors to carry out a contact tracing study, testing stool samples from family members that lived in the same building as the child for SARS-CoV-2 RNA; they found all the family members to be asymptomatic and positive for viral RNA in their stool. Notably, the child’s male parent (am1) continued to have extended shedding of viral RNA out to 90 days from the start of treatment. The authors collected stool samples from this parent to understand the pathobiology of SARS-CoV-2. Specifically, they expand on their previous observation (featuring two shared authors with the current manuscript) that this virus could potentially display bacteriophage-like behavior and infect bacteria. In this previous work, they carried out in vitro culturing assays and reported that the concentration of viral RNA increased with time when incubated with fecal bacteria 1 . In another publication (featuring seven shared authors with the current manuscript), the authors use TEM and immunofluorescence microscopy and report visualizing SARS-CoV-2 human fecal-oral bacteria” can infect bacteria a bacteriophage. a symptomatic 21-month-old nasopharyngeal sample tested negative on a rapid antigen test, but feces tested positive for SARS-CoV-2 viral RNA on a Luminex assay. This led the authors to carry out a contact tracing study, testing stool samples from family members that lived in the same building as the child for SARS-CoV-2 RNA; they found all the family members to be asymptomatic and positive for viral RNA in their stool. Notably, the child’s male parent (am1) to extended shedding of viral RNA out to 90 days from the start of treatment. The collected stool samples from this parent to understand the pathobiology of SARS-CoV-2.


Introduction
In the past two years, humanity has been combating the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a positive, single-stranded RNA virus of the Coronaviridae family, specifically of the subfamily Orthocoronavirinae (usually called "coronaviruses"). Its closest known relatives are those found in bat feces, like the coronavirus RaTG13. 1 Xu et al. (2020) 2 studied viral behavior in 10 children, ranging in age from two months to 15 years. Although all of them were positive to the initial nasopharyngeal test, for eight of them, the viral charge was also positive in the stool. Moreover, they continued to test positive in the stool even after the negative nasal swab for several days after hospital discharge. In another Chinese study, the researchers found viral positivity in the fecal samples of 205 patients. 4 Many studies [3][4][5] have observed that fecal-oral transmission of the virus is possible and that it is very common to detect this virus in feces. Nevertheless, in comparison to the closest SARS-like viruses, SARS-CoV-2 appears to diverge in the receptor-binding domain of the spike glycoprotein, which is considered a key player in the entrance of the virus in human eukaryotic cells throughout its interaction with the angiotensin-converting enzyme 2 receptor (ACE-2), which in turn is considered the entry point of the virus. 6 ACE-2 receptors and host cell transmembrane serine protease 2 (TMPRSS2) are abundant throughout the intestinal tract 7,8 and several studies have reported altered intestinal bacterial flora or intestinal bacterial co-infection in COVID-19 patients. [9][10][11] In terms of hosts, coronaviridae members are neither humanspecific nor new in terms of discovery and treatments: a recent review describes the numerous zoonoses caused by the Coronaviridae family members, 12 and scientists searched for the pathogen in the stool, 13 a method that was, and continues to be, very common in the veterinary field. Among the coronaviruses previously found and analyzed in feces, there are those responsible for animal diseases like the calves' enzootic pneumonia (caused by Bovine coronavirus, BCoV), or the porcine epidemic diarrhea (caused by the Porcine Epidemic Diarrhea Virus, PEDV). These diseases and other coronavirus-related ones very often show as initial clinical manifestation of violent diarrhea, and the affected animals have a significant alteration of the intestinal mucosa. 12,14,15 Observations of possible links between the animal gut microbial environment and coronaviruses have been reported in some studies, 16-20 supported also by the use of transmission electron microscopy (TEM) image analysis which screens and looks for viruses-like particles. 8,21 The observation of SARS-CoV-2 particles by TEM can complement the molecular traces of it. 22 Finally, it is worth noting that almost all of the latest characterized SARS-like viruses have been found and sequenced in bat fecal samples. 23 Here, we report the case of a symptomatic child whose family members had negative results with rapid antigen nasopharyngeal swab test. Analyses of fecal samples detect the viral RNA presence in the feces of the child and of all her relatives, which thus resulted to be positive asymptomatic. Microscope image analyses confirm the presence of SARS-CoV-2-like particles on fecal samples of the family and suggest that bacteria, reservoirs of the virus, are the most critical factors of fecal-oral transmission in this pandemic. The present case report also emphasizes the importance of the rapid detection of SARS-CoV-2 in symptomatic and non-symptomatic subjects with negative results from nasal and oropharyngeal swabs by analyzing stool samples, and emphasizes the importance of the bacteriophagic mechanism of the virus and its fecal-oral transmission.

Case series description
A 21-month-old female, Caucasian child, presented to us with severe bilateral conjunctivitis, conspicuous lacrimation, diarrhea, malodorous stools, restlessness, and fever (38°C). The child's medical history was negative for any disease. Parents reported that about a year earlier, she had a period when she had a severe cold. They were alarmed by violent

REVISED Amendments from Version 1
The second version of the manuscript has some changes. The title has been changed to "The first report on detecting SARS-CoV-2 inside bacteria of the human gut microbiome: A case series on asymptomatic family members and a child with COVID-19". The words "bacteria of the human gut microbiome" had more appropriate to the written context. Having word limits for a case series, according to the guidelines, we added the supplementary materials (s.m.) to provide more details to the readers without requiring them to find references in our already published articles. Supplementary materials can be viewed, in accordance with the journal guidelines, at the following DOI: 10.5281/zenodo. 6974414. Few words or phrases have been improved for greater clarity. Two new control panels have been added, Figure 3, panels F and G, in tiff format, 300 dpi, RGB, showing additional control in the feces of a healthy subject. Table 1 has been expanded with a column showing the results of nasopharyngeal antigen swabs. References 9-11 and 16-20 have been added. Reference 48 is now correct and had been described in the text (v1). Dr. Vincenzo Costanzo, who acquired the images of the control samples in Figure 3, was added in the acknowledgments. All authors approved the changes and additions. diarrhea, which was preceded by 24 hours of constipation, as well as by the abnormal bilateral conjunctivitis with uncontrollable lacrimation. Rapid blood tests showed the following values (in bold are those out of normal range, NR): creatinine 0.18 mg/dL (NR: 0.40-1.10 mg/dL); glucose 97 mg/dL (NR: 60-110 mg/dL); aspartate transaminase 45 I.U. (NR: 10-50 I.U.); alanine transaminase 28 I.U. (NR: 10-35 I.U.); sodium 139 mEq/L (NR: 136-150 mEq/L); potassium 5.82 mEq/L (NR: 3.50-5.10 mEq/L); chloride 95 mEq/L (NR: 98-107 mEq/L); calcium 5.50 mEq/L (NR: 4.25-5.25 mEq/L); C-reactive protein 2.60 mg/L (NR: 0-5 mg/L); iron 28 mcg/dL (NR: 59-158 mcg/dL). Other complete blood count values were in the normal range.
The Caucasian family (six adults, three children) came to us, in the autumn of 2020, during one of the Italian regional lockdown periods. Some specific information on the family members were recorded, including age, sex, medical history, occupations, and relationships (see Table 1). They live in close proximity, divided among three apartments in one building (Figure 1 panel A). The parents reported that the children never had a babysitter since this task was entrusted to their grandparents, who were in their building. Moreover, they reported that since the outbreak of the pandemic (March 2020), they had adopted a series of measures, probably excessive in their opinion, with the purpose of protecting the grandparents and children from sickness. Such measures included no contact with people outside the family context, disinfection of every product purchased, no summer holidays, no eating at restaurants or other public places, and limited outings for the four parents (am1, af1, am3, af3) for work reasons only. The grandfather (am2), grandmother (af2), and the three children (cf1, 2cf1, cm3) did not leave the building for the duration of the lockdown ( Figure 1A and Table 1). All the parents (am1, af1, am3, af3) of the children working in the health care sub-area left home daily to work, and one of them worked in another geographical region. Considering their work position it is most likely that the family infection started with the contagiousness of one of the four parents (am1, af1, am3, af3) who were asymptomatic during working hours. Of interests is the medical history of one adult (am1), the father of child cf1 (our COVID-19 patient), that was hospitalized precisely one year prior (autumn 2019) with escalating symptoms of violent diarrhea, abdominal pain, fever (38°C), dyspnea, cough, headache, shortness of breath, and fainting. There was saturation of 91 SpO2%, right bundle branch block, increased D-Dimer, increased liver values (GOT and GPT), and mild lymphopenia, treated with antibiotics.
We initially performed rapid antigen nasopharyngeal swab test (COVID-19 Ag Rapid Test Device, Abbott 41FK10) on the child (cf1), and it was negative. The same test was also performed on the parents (am1, af1) and the other six family members, and all results were negative. We had, in line with previous studies, 24,25 experience of multiple negative results SARS-CoV-2 real-time reverse transcriptase polymerase chain reaction (RT-PCR) tests on oropharyngeal/nasopharyngeal (OP/NP) swab samples from individuals with a strong clinical suspicion of COVID-19. 26 Being in the presence of a very young patient, it was decided to adopt a fast high-throughput COVID-19 screening approach to detect the presence of SARS-CoV-2 directly from stool samples: in the following 24 hours, stool samples were collected from all nine family members, and molecular testing for SARS-CoV-2 was performed by using Luminex technology 27,28 as described by us previously. 29 Negative and positive controls as bacterial cell cultures of stool samples were those used and described in this previous study. 29 A summary of the analyses is reported (all methods and materials are detailed in supplementary materials s.m.) in Figure 1C-D and Table 1 52 : all family members had positive results to the Luminex molecular test, and the child with symptoms (cf1) showed the highest value of the Luminex assay. The other family members did not manifest any symptoms, despite being positive for the presence of viral RNA in their stools.
The child was treated for 48 hours only with rehydration and probiotics only; because of the absence of significant symptoms such as cough or dyspnea, no cortisone or antibiotics were administered. Conjunctivitis and lacrimation ceased about 72 hours later and the patient was discharged. The entire family, including the reported patient, were then instructed to take probiotics (Lactobacillus reuteri, 100 million units, one time per day, and Bacillus clausii 2 billion units, per day) in addition to bromelain, 300 mgr. per day, and colloidal copper, 20 ppm (parts per million) per day for 30 days, only as rebalancers of bacterial flora. After 60 days, both the rapid antigen nasopharyngeal swab test (COVID-19 Ag Rapid Test Device, Abbot 41FK10) and the Luminex test were repeated: all family members were negative to the rapid antigen tests, and only one family member ( Figure 1D -am1) continued to have Luminex positive results. Patient am1, male, Caucasian and a healthcare employee, continued the treatment until he became negative at day 90 for the presence of SARS-CoV-2 in stools. The feces of this patient was cultured in bacterial culture media and after 30 days, the pellet of bacteria, have been analyzed by TEM, immune-EM, and by fluorescence microscopy, and a set of obtained images is shown in Figure 2 (for more details see supplementary material-s.m.). 52 At day 30 of bacterial culture of feces patient am1, the Luminex molecular test confirmed the presence of SARS-CoV-2 and the RNA viral conentration was increased from 24 arbitrary unit (AU) (initial) to 520 AU (Final) ( Figure 1B) in accordance with our previous observations. 29 Transmission electron microscope images (panels A and B of Figure 2-Tecnai G2 Spirit BioTwin; FEI, equipped with a VELETTA CCD digital camera -Soft Imaging Systems GmbH) SARS-CoV-2 (black arrows) inside a bacterium (A) and outside a matrix  resembling extracellular lysate of a bacterium (B). No eukaryotic cells have been ever observed after 30 days of bacterial culture. Post-embedding immunogold ( Figure 2 Panel C, D): bacteria pellets were fixed with a mixture of 0.05% glutaraldehyde of 4% paraformaldehyde in 0.1M PBS (Phosphate-buffered saline) buffer, washed in PBS buffer, pelleted at 10000g and included in 3% agarose. The agarose block was cut into tissue-size pieces and the slices were post-fixed in 2% OsO 4 , dehydrated in a series of ethanol solutions of increasing concentration and in propylene oxide and finally embedded in Epon 812. Thin sections were cut from embedded specimens using Reichert Jung Ultra microtome and are applied to Formvar/Carbon Supported nickel grids. Sections were blocked with normal goat serum for 1h at room temperature, incubated with rabbit monoclonal to SARS-CoV-2 nucleocapsid protein antibody (EPR24334-118, Abcam) and then with secondary anti-rabbit antibody 10nm gold-conjugated (Aurion). 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).
The immunofluorescence microscope ( In one of the first studies on SARS-CoV-2 in Wuhan, prominent symptoms of COVID-19 patients are described, including diarrhea 35 and in children, gastrointestinal disorders are the most prevalent. 32 The persistence of coronaviruses in feces, for a long time, had already been observed many years ago. In one of the first case reports of 1982, Baker et al. 36 described the case of a 47-year-old Indian man who underwent surgery for a duodenal ulcer when he was 13 years old. The symptoms that forced hospitalization were diarrhea and steatorrhea. The man was monitored for eight months, and in 17 fecal samples, coronavirus-like particles were observed by electron microscopy. The images show two ovoid/geoid shaped coronavirus particles with the spike protein evident and one circular shaped coronavirus particle but without surface proteins, like those here reported in Figure 2.
Inclusion of symptoms other than respiratory, such as gastrointestinal symptoms, seems to be very important in the diagnostic process. Although diarrhea and conjunctivitis with lacrimation, as in our case, may be unlinked, they can be related to each other if the gut microbiota and the central, peripheral, and autonomous nervous systems are taken into account. The gut microbiota 37 seems to be extremely important and interconnected with the central, peripheral, autonomic, neuroimmune, and neuroendocrine nervous system axis. An altered gut microbiota or the total absence of bacteria, as in germ-free mice, can affect areas of the brain, including the hippocampus, the point of end of olfactory system. 38 Several studies have reported an impairment of intestinal gut microbiota 39 or respiratory and intestinal bacterial coinfection in  As shown in Figures 2-3, bacteria could play crucial role in the possibility of fecal-oral transmission. This news isn't so far away from the most recent studies 29 in which we described that RNA replication of the SARS-CoV-2 virus can take place in bacterial cultures. We also described that the use of certain drugs can decrease its replication in vitro.
Moreover, in the same work, we observed, by mass spectrometry, the mutational phenomenon of viral proteins in bacterial cultures. Other authors have also noted the possibility that the spike protein of the SARS-CoV-2 may interact with the lipopolysaccharide of Escherichia coli 41 or that the absence of proteobacteria could play a key role in the pathogenesis of respiratory viral diseases. 38 This is why early localization in the stool assumes considerable importance. Since the discovery of SARS-CoV-2, a plethora of commercial tests have become available, and, currently, more than 1,700 tests are commercialized in the European Union countries (source JRC COVID-19 In Vitro Diagnostic Devices and Test Methods Database 42 ). Rapid Antigen Tests (RATs) are recommended to be routinely used, 43,44 especially on oropharyngeal/nasopharyngeal (OP/NP) swab samples. Researchers have had sometimes problems in terms of sensitivity and specificity with some of them. 45 Problems may arise because the tests were initially evaluated on samples from patients with severe COVID-19, who are suggested to develop a much higher immune response than those with mild or asymptomatic disease. 46 RT-PCR is considered the gold standard method for detection of SARS-CoV-2. However, we had previous experience of multiple negative results SARS-CoV-2 RT-PCR tests on OP/NP swab samples from individuals with a strong clinical suspicion of COVID-19. 26 Mardian et al. 2021 recommend fecal detection of viral RNA when nasopharyngeal swab data are questionable. 47 "In a Systematic Review and Meta-analysis, at the beginning of the pandemic, it was observed that viral RNA was present in the stool in 48.1% of patients during the disease and that 70.3% of patients had prolonged shedding that could extend beyond 33 days from the onset of the disease." 48 Finally, in a recent study aimed to evaluate the role of fecal-oral transmission, unique RNA SARS-CoV-2 genomic sequence mutations have been observed by performing next-generation sequencing on the fecal samples. 25 In this case the Luminex technology as molecular testing tool was chosen because it is ideal for fast high-throughput COVID-19 screening and its clinical performance have been evaluated. 49 In consideration that SARS-CoV-2 was detected at low levels in fecal samples, 50 in addition to molecular test, was agreed to verify the presence of the virus by acquiring images of at least one sample. As proposed by Dittmayer and colleagues,22 in the case of COVID-19 diagnosis, the use of image analysis to confirm the presence of SARS-CoV-2 particles complements the detection of molecular traces of SARS-CoV-2 specific proteins or nucleic acids (and vice versa). In studies of infectious diseases, TEM is used very often to definitively prove the presence of an infectious unit. The images were obtained by TEM, immune-EM, and by fluorescence microscope. What we have noted is (in agreement with our first observations 29 ), that could be present an important role of bacteria in the fecal-oral transmission of SARS-CoV-2.
The only limitations of such investigations are the high costs and long waiting times.

Conclusions
Here we report the case of a child symptomatic for COVID 19, transmitted by one of the parents, whose relatives had tested negative on the rapid antigenic nasopharyngeal swab test. Analyses of fecal samples by high-throughput COVID-19 screening (Luminex technology) allowed us to accurately detect the viral RNA presence in the faces of the child and of all her relatives, which thus resulted to be positive asymptomatic.
Microscopy images analysis was used as complementary approach to confirm the presence of SARS-CoV-2 in bacterial cultures obtained by fecal sample of an infected individual with the viral RNA load positive individual. The images obtained by TEM, immune-EM and by fluorescence microscope show SARS-CoV-2 inside human gut bacteria and outside a matrix resembling extracellular bacterial lysates, in agreement with a bacteriophage mechanism. 29 This first observation invites us to pay more attention to the fecal-oral transmission route of the virus and suggests as a further possible reservoir of the virus also the bacteria of the human gut microbiome.
We believe that accurate analysis of the human gut microbiome during viral infections, including SARS-CoV-2 infections, may be of great importance and may aid in diagnosis when other tests fail. 26 According to the other studies 47 faster and more versatile tests should be improved to decrease or cope with the contagiousness of the pathogens, especially to detect them in the stools. The observation of all clinical symptoms, typically respiratory, gastrointestinal, and neurological, combined with molecular testing (stool, sputum, tear, other fluids) and image analysis, represents the key for understanding the interaction of SARS-CoV-2 with the human gut microbiome and its product. 51 Therefore, for the provision of the correct epidemiology, diagnosis and accurate therapeutic approach is important in the treatment of COVID-19, especially in challenging cases, such as the one reported here. This case also highlights the possibility of contagion from asymptomatic parents to their children.

Data availability
All data underlying the results are available as part of the article and are viewable at the following DOI https://doi.org/ 10.5281/zenodo.6974414, according to the journal guidelines; https://zenodo.org/record/6974414#.YvFp6-xBxmA.

Underlying data
All data underlying the results are available as part of the article and no additional source data are required.

Extended data
Zenodo: Supplementary materials (s.m.) of "The first report on detecting SARS-CoV-2 inside bacteria of the human gut microbiome: A case series on asymptomatic family members and a child with COVID-19" https://doi.org/10.5281/ zenodo.6974413.
This project contains the following extended data: -Supplementary material: Materials and methods of the tests described in the paper (detection of viral RNA by Luminex method, immunofluorescence at microscopy, electron microscopy, proteomics, and viral protein labeling by nitrogen radioisotope. Data are available under the terms of the Creative Commons Attribution 4.0 International (CC BY Creative Commons 4.0 license).

Consent
Written informed consent for publication of their clinical details and clinical images was obtained from the parents of the child. Written informed consent for publication of their clinical details and clinical images was also obtained from all other patients involved in the study.  This manuscript by Brogna C et al. reports the first observation of SARS-CoV-2 "inside human fecaloral bacteria" suggesting that SARS-CoV-2 can infect bacteria like a bacteriophage. This study is inspired by a symptomatic 21-month-old child whose nasopharyngeal sample tested negative on a rapid antigen test, but feces tested positive for SARS-CoV-2 viral RNA on a Luminex assay. This led the authors to carry out a contact tracing study, testing stool samples from family members that lived in the same building as the child for SARS-CoV-2 RNA; they found all the family members to be asymptomatic and positive for viral RNA in their stool. Notably, the child's male parent (am1) continued to have extended shedding of viral RNA out to 90 days from the start of treatment. The authors collected stool samples from this parent to understand the pathobiology of SARS-CoV-2.
Specifically, they expand on their previous observation (featuring two shared authors with the current manuscript) that this virus could potentially display bacteriophage-like behavior and infect bacteria. In this previous work, they carried out in vitro culturing assays and reported that the concentration of viral RNA increased with time when incubated with fecal bacteria 1 . In another publication (featuring seven shared authors with the current manuscript), the authors use TEM and immunofluorescence microscopy and report visualizing SARS-CoV-2 particles in gut bacteria 2 .
In the current work, they feature these same in vitro and microscopy experiments with a different stool sample and report the same conclusion -that bacteria in the gut are infected with SARS-CoV-2 viral particles.
I recommend this manuscript be rejected in its current form. It may be sent out for reviews again pending major revisions.

Major suggestions:
Throughout the manuscript, the authors suggest the fecal-oral transmission of SARS-CoV-2. However, whether there are infectious viral particles in stool capable of being transmitted is 1.
at best still debated, with more evidence to the contrary 3 . Especially given the relevance of this matter to clinical decisions and public health, I encourage the authors to present a balanced view.
The authors report viral RNA concentration determined through Luminex in arbitrary units (AU). However, my understanding is that Luminex can be set up to contain standards that reveal an absolute concentration of viral RNA. Given how viral RNA concentrations are central data to the conclusions in this work, I believe that reporting the absolute concentration of viral RNA is important. Additionally, this will make the observations here more replicable across labs and viral RNA detection techniques.

2.
Regarding the reported viral RNA concentrations, it is unclear what the specificity, sensitivity, and detection range of the Luminex assay are as carried out by the authors. Therefore, I recommend the authors include relevant controls to estimate these. Reporting this information along with their experiments will add important validity and context to the data. 3.

4.
The authors conclude from the microscopy data that fecal bacteria are infected by SARS-CoV-2 in samples collected on day 30 from an adult patient, am1. Notably, this patient along with the others in the study was prescribed probiotics with two Gram-positive bacteria ( Lactobacillus reuteri and Bacillus clausii) in their diets for 30 days prior to sample collection. In the event that bacteria are in fact infected by SARS-CoV-2, it is unclear if this is an artefact of the probiotic supplements and treatment regime.

5.
I encourage the authors to provide more methodological information in the manuscript. As a matter of principle, manuscripts should in and of themselves provide sufficient information for readers to repeat experiments without having to go down the rabbit hole of chasing down references to other works. The current manuscript exhibits this concern that is prevalent in scientific publications. Some instances of this in the current manuscript include -"molecular testing for SARS-CoV-2 was performed by using Luminex technology as described by us previously" -It is unclear how the samples were prepared, if controls were included and what the parameters of this assay were.
○ Methodology used to isolate and culture bacteria from stool -It is unclear how bacteria were isolated from stool. This is important information to get a sense of how rigorous this method was, whether there are inherent biases regarding what bacteria are favored in the culturing process and if there are chances for other residual contaminants from the fecal sample.
○ Methodology used to collect and preserve stool samples -the majority of existing reports are unable to culture viable SARS-CoV-2 from fecal samples, even when the samples are preserved in Viral Transport Media (VTM). Therefore, clarity about how the current work collected and preserved samples are crucial to understanding how they have been successful at recovering viable viral particles.

Minor suggestions:
Request citation and clarity on the statement -"several studies have reported….intestinal bacterial co-infection in COVID-19 patients".

1.
Request citation and clarity on the statement -"Observations of possible links between the animal gut microbial environment and coronaviruses have been reported over time". The current citations do not have evidence for a possible link to the gut microbial environment. 2.

Fig 1 reports "Viral load"
although what is actually reported is the viral RNA concentration, which can be different from viral load because not all viral particles are lysed and provide RNA for detection, and some viral RNA is reported to come from non-viral reservoirs 4 .

3.
The manuscript calls for major editorial revisions given numerous improper usages of phrases. For instance -a) There are no "fecal-oral bacteria", which suggests specific bacteria in the feces are able to be transmitted orally. b) It is unclear what "Bacterial feces" means. c) "RNA viral load count" appears miswritten.

4.
Additional notes: I don't have experience with interpreting TEM images and therefore leave this comment here rather than as a cause for major revision. TEM images are complex requiring the appropriate collection and processing of samples, the inclusion of controls, and careful interpretation of observations. The improper use of EM to study SARS-CoV-2 from tissue samples has been a cause for concern 5,6 . To my untrained eye , Fig 2 seems to be another example of misinterpreted EM data because - If Fig 2a in fact displays SARS-CoV-2 viral particles inside a bacterium, it is surprising to me that the bacterial host appears to be entirely filled by viral particles with no room for its essential, native molecules. To me, this figure seems like that of the widely described multivesicular bodies that are unrelated to SARS-CoV-2 and yet regularly misinterpreted 6 .  The hypothesis that SARS-CoV-2 may infect bacteria is both biologically fascinating and clinically highly relevant. This is because there are no examples that I know of or can find of viruses that affect humans that can also retain the molecular machinery to affect bacterial cells. Therefore, the idea that this is possible is biologically fascinating and can pave the path to many follow-up studies. Further, SARS-CoV-2 continues to be a major threat, having claimed over 6 million people worldwide. Therefore, careful evaluation of the potential sources of future outbreaks is critical from a personal and public health standpoint. If in fact 2.
bacteria in the gut can harbor infectious SARS-CoV-2 viruses, this adds to another dimension of the COVID-19 pandemic that calls for urgent precautionary measures. Unfortunately, the current manuscript is missing many controls and methodological details, and has insufficient data to make the case that bacteria are in fact infected by SARS-CoV-2. Given the hugely significant impact that concluding gut bacteria can be infected by SARS-

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
Author This manuscript by Brogna C et al. reports the first observation of SARS-CoV-2 "inside human fecal-oral bacteria" suggesting that SARS-CoV-2 can infect bacteria like a bacteriophage. This study is inspired by a symptomatic 21-month-old child whose nasopharyngeal sample tested negative on a rapid antigen test, but feces tested positive for SARS-CoV-2 viral RNA on a Luminex assay. This led the authors to carry out a contact tracing study, testing stool samples from family members that lived in the same building as the child for SARS-CoV-2 RNA; they found all the family members to be asymptomatic and positive for viral RNA in their stool. Notably, the child's male parent (am1) continued to have extended shedding of viral RNA out to 90 days from the start of treatment. The authors collected stool samples from this parent to understand the pathobiology of SARS-CoV-2.
Specifically, they expand on their previous observation (featuring two shared authors with the current manuscript) that this virus could potentially display bacteriophage-like behavior and infect bacteria. In this previous work, they carried out in vitro culturing assays and reported that the concentration of viral RNA increased with time when incubated with fecal bacteria1. In another publication (featuring seven shared authors with the current manuscript), the authors use TEM and immunofluorescence microscopy and report visualizing SARS-CoV-2 particles in gut bacteria2. In the current work, they feature these same in vitro and microscopy experiments with a different stool sample and report the same conclusion -that bacteria in the gut are infected with SARS-CoV-2 viral particles.
I recommend this manuscript be rejected in its current form. It may be sent out for reviews again pending major revisions.

Major suggestions:
Throughout the manuscript, the authors suggest the fecal-oral transmission of SARS-CoV-2. However, whether there are infectious viral particles in stool capable of being transmitted is at best still debated, with more evidence to the contrary3. Especially given the relevance of this matter to clinical decisions and public health, I encourage the authors to present a balanced view.
R: First, we are honored by the reviewer's comments on the fecal-oral route of transmission od the virus SARS-CoV-2 because they were our doubts at the beginning of the pandemic and were the reason we wanted to do the check on prokaryotic cells.

In fact, we found it really embarrassing that no major research group in the world has documented a positive or also negative control on the interaction between Coronavirus, in general in the last 30 years, and SARS-CoV-2 in the last 2 years and prokaryotic unicellular cells.
Only the famous diatribes on the occurrence of bacterial cofactor in HIV (RNA virus) infection can be found in the literature. We respect the reviewer's opinion on the epidemiology and transmission route of the virus, but we believe that in a balanced context, the oro-fecal route of transmission should also, and not only be considered. However, the reviewer pays attention to this issue, and in line with his opinion, we have modified the text by adding the word "also" or "possible" next to "transmission route" where this was possible.

Reference 3 that the reviewer cites is a letter, a commentary by
The authors report viral RNA concentration determined through Luminex in arbitrary units (AU). However, my understanding is that Luminex can be set up to contain standards that reveal an absolute concentration of viral RNA. Given how viral RNA concentrations are central data to the conclusions in this work, I believe that reporting the absolute concentration of viral RNA is important. Additionally, this will make the observations here more replicable across labs and viral RNA detection techniques. Regarding the reported viral RNA concentrations, it is unclear what the specificity, sensitivity, and detection range of the Luminex assay are as carried out by the authors. Therefore, I recommend the authors include relevant controls to estimate these. Reporting this information along with their experiments will add important validity and context to the data.

. and panels F and G show a group of gram+ bacteria by fluorescence, derived from the stool bacteria culture of a healthy 18-month-old child (with healthy parents and never ill with SARS-CoV-2 at the time of collection and with and with their written consent) negative to molecular test to SARS-CoV-2, although the other primary antibody to the nucleocapsid protein is also included and does not show a red signal". Line 193-199
The authors conclude from the microscopy data that fecal bacteria are infected by SARS-CoV-2 in samples collected on day 30 from an adult patient, am1. Notably, this patient, along with the others in the study was prescribed probiotics with two Gram-positive bacteria (Lactobacillus reuteri and Bacillus clausii) in their diets for 30 days prior to sample collection. In the event that bacteria are in fact infected by SARS-CoV-2, it is unclear if this is an artefact of the probiotic supplements and treatment regime. I encourage the authors to provide more methodological information in the manuscript. As a matter of principle, manuscripts should in and of themselves provide sufficient information for readers to repeat experiments without having to go down the rabbit hole of chasing down references to other works. The current manuscript exhibits this concern that is prevalent in scientific publications. Some instances of this in the current manuscript include -"molecular testing for SARS-CoV-2 was performed by using Luminex technology as described by us previously" -It is unclear how the samples were prepared, if controls were included and what the parameters of this assay were. Methodology used to isolate and culture bacteria from stool -It is unclear how bacteria were isolated from stool. This is important information to get a sense of how rigorous this method was, whether there are inherent biases regarding what bacteria are favored in the culturing process and if there are chances for other residual contaminants from the fecal sample. Methodology used to collect and preserve stool samples -the majority of existing reports are unable to culture viable SARS-CoV-2 from fecal samples, even when the samples are preserved in Viral Transport Media (VTM). Therefore, clarity about how the current work collected and preserved samples are crucial to understanding how they have been successful at recovering viable viral particles.
R: We thank the reviewer for this valuable advice and apologize because it was not our intention to direct him to read our other work, but unfortunately, as the manuscript is set up as a case series, we had a predetermined number of words and checklist guidelines to follow. To overcome this problem, we suggested that he read our other papers, but the reviewer rightly asked for more details, and we have summarized the

support that SARS-CoV-2 genomic and subgenomic RNAs are present in diagnostic samples even in late infection/after active infection' and 'The detection of subgenomic RNA is therefore not direct evidence of active infection instead its presence at lower levels than virion genomic RNA results in detection for a shorter period of time unless using, e.g., highly sensitive NGS'.
Similarly, in the tables presented in figure 1, we do not want to indicate who among the family members in the presented case series is infectious or not because the story in the paper describes well that they are all asymptomatic, except for the little girl. In the paper, we never speculate on the correlation between viral charge and high infectivity. The tables only show how with the passage of a fairly long time (figure 1 C and D), 60 days, the presence of traces of viral RNA is no longer detected, in agreement with many contemporary studies. It should also be pointed out that reference 4, Alexandersen et al., describes the nasopharyngeal sampling as a viral RNA collection event, and readers and scientists should reflect that the sampling is performed on mucous membranes that are overabundant with bacteria and that it cannot be excluded that subgenomic sequences in non-SARS-CoV-2 sufferers could have bacteria, also, as a reservoir.
We do not associate the viral load to the disease state, nor do we speculate on high viral replication and high infectivity. Viral load is now a usual term used in scientific work, and the accuracy of the reviewer is relevant, but at this point, should specify the wording of many of the works currently published. However, we corrected everywhere the legend of figure 1 and other phrases in "RNA viral Concentration." The manuscript calls for major editorial revisions given numerous improper usages of phrases. For instance -a) There are no "fecal-oral bacteria", which suggests specific bacteria in the feces are able to be transmitted orally. b) It is unclear what "Bacterial feces" means. c) "RNA viral load count" appears miswritten. Figure 2 ( for more details see supplementary material-s.m.)». Line 133-136 c) The word count is a typo, and we followed the reviewer's suggestion and pointed out that it is RNA concentration Additional notes:

R : a) we apologize to the reviewer. We want to say bacteria from the gastrointestinal tract, and we replaced the phrase with "bacteria of the human gut microbiome." b) The sentence was rewritten: « The feces of this patient was cultured in bacterial culture media and after 30 days, the pellet of bacteria, have been analyzed by TEM, immune-EM, and by fluorescence microscopy, and a set of obtained images is shown in
I don't have experience with interpreting TEM images and therefore leave this comment here rather than as a cause for major revision. TEM images are complex requiring the appropriate collection and processing of samples, the inclusion of controls, and careful interpretation of observations. The improper use of EM to study SARS-CoV-2 from tissue samples has been a cause for concern5,6. To my untrained eye, Fig 2 seems to be another example of misinterpreted EM data because - If Fig 2a in fact displays SARS-CoV-2 viral particles inside a bacterium, it is surprising to me that the bacterial host appears to be entirely filled by viral particles with no room for its essential, native molecules. To me, this figure seems like that of the widely described multivesicular bodies that are unrelated to SARS-CoV-2 and yet regularly misinterpreted6. The figures don't appear to have sufficient resolution to highlight the ultrastructures typical of SARS-CoV-2 that are required to unmistakable identify viral particles5. If Fig 2b in fact presents the case of a bacteria with viral particles around it, it merely appears like viral particles proximal to the bacteria in this image, with no evidence that these viruses in fact infected the bacteria. The conclusion that viral particles are found in the extracellular matrix of gut bacteria is an over-reach without sufficient evidence.  Figures 1C and 2C, are clearly visible viral particles indicated by the authors as coronavirus particles perfectly identical to those we show inside the bacterium in our Figure 2A and around the lysed bacterium in Figure 2B. Moreover, the authors cite the following sentence to demonstrate that the protein spike, unless a special preparation with tannic acid is used, is almost never visible in microscopic images as they report: We remind the reviewer that these are the first images of coronavirus in bacterial cultures. There are no other comparisons in the current literature, so any speculation on the present manuscript finds as debate the serious shortcoming of the failure to control infectivity first on prokaryotic cells, which are much more abundant above the epithelial layer where virus-receptor interaction takes place. What should be done before proclaiming for certain and establishing some possible pathogenetic mechanisms of infectivity, i.e., some possible viral protein and epithelial cell receptor binding interactions, is the control between coronaviruses in particular but also other RNA viruses in general and bacterial cells, much more abundant, it bears repeating, within the human microbiome. Moreover, even if an interaction does not take place, control also over bacterial metabolism and what they produce (2,14) in the presence of a novel viral pathogen should in no way be excluded. This, too, is a serious deficiency in possible controls.
The hypothesis that SARS-CoV-2 may infect bacteria is both biologically fascinating and clinically highly relevant.
R: We apologize to the reviewer: but with due respect: it is no longer a hypothesis but new observations (ref 2,14, 15).
We thank the reviewer for giving us an opportunity to engage in constructive scientific debate and to improve the present paper by highlighting what he thought was right and by improving what he thought was appropriate.
This is because there are no examples that I know of or can find of viruses that affect humans that can also retain the molecular machinery to affect bacterial cells. Therefore, the idea that this is possible is biologically fascinating and can pave the path to many follow-up studies. Further, SARS-CoV-2 continues to be a major threat, having claimed over 6 million people worldwide. Therefore, careful evaluation of the potential sources of future outbreaks is critical from a personal and public health standpoint. If in fact bacteria in the gut can harbor infectious SARS-CoV-2 viruses, this adds to another dimension of the COVID-19 pandemic that calls for urgent precautionary measures. Unfortunately, the current manuscript is missing many controls and methodological details, and has insufficient data to make the case that bacteria are in fact infected by SARS-CoV-2. Given the hugely significant impact that concluding gut bacteria can be infected by SARS-CoV-2 through a bacteriophage-like property can have to humanity, I encourage the authors and editorial team to exercise caution and responsibility in promoting this conclusion with insufficient evidence.
R : We believe that the reviewer now has much more clarification, but may notice more information about the concepts laid out and the importance of doing the checks between viruses and bacteria, to avoid epidemiologically and pathogenic misdiagnoses in the article under review also on F1000- This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reviewer comments -
The Figure 1B legend needs to be properly written or displayed. 1. Figure 1C, correct the spelling of culture as in Bacteria culture (legend). 2.
In Table 1, data pertaining to the nasopharyngeal swab test need to be presented for all the subjects.

3.
"At day 30 of bacterial culture of feces patient am1, the Luminex molecular test confirmed the presence of SARS-CoV-2 and the RNA viral load count was increased from 24 arbitrary unit (AU) (initial) to 520 AU (Final) ( Figure 1B)..." -The figure description needs to be better ( Figure 1B). The authors should better describe whose fecal sample was used in the culture in the figure description for 1B.

4.
"Bacterial feces of this patient, after 30 days of bacterial culture, have been analyzed by TEM, immune-EM, and by fluorescence microscopy, and a set of obtained images is shown..." -Instead of bacterial feces, change to feces of this patient was cultured in bacterial culture media. Figure 2D is not clear to this reviewer. 6.

5.
"In addition, the RNA virus could be present in the 48,1% of patients who were negative to OP/NP swab tests until 33 days." -Please correct the % depiction.

7.
"Here, we report the case of a symptomatic child for COVID 19, brought to her by one of the parents, whose family members had negative results with rapid antigen nasopharyngeal swab test." -Please correct the grammatical error.

8.
This reviewer has reservation on the "fecal-oral" transmission route being used by the authors. This paper and the earlier paper on this topic by the authors showed possible replication of the SARS-Cov2 virus like particles in bacterial culture. But neither observations prove the route of the viruses coming into the feces. As far as this reviewer is concerned, we still do not know how the virus gets to the gastrointestinal tract. So, instead of "fecal-oral" bacteria, gastrointestinal bacteria may be written.

9.
"This surprising finding allows us to better clarify the first fecal-oral transmission of the virus and clearly shows that the reservoir of the virus is neither adults nor children but simply bacteria." -This reviewer does not agree with this statement especially the second half of it.

10.
Overall, this is an interesting finding and corroborates the earlier report of the same author (F1000Res. 2021 May 11; 10:370) about the presence of the SARS-Cov2 like virus particles in bacterial culture. This report is an advancement as some evidence of the presence of the virus like particles have been shown using TEM, immune-fluorescence microscopy. The presence of SARS-Cov2 in feces has been documented before and as such this report does not add anything new to this however what is interesting is the replication potential of the virus in bacterial culture. The authors might want to provide more assays and evidence to showcase the "phage-like" activity of the SARS-Cov2 as they propose.
Finally, please crosscheck the text. Lots of grammatical and contextual errors are found, some of which have been highlighted above.
fully revised version of the manuscript.
Best regards, Carlo Brogna, on behalf of the authors. represent a potential reservoir of the virus and, as in the work cited above, we argued that it could behave as both a lytic and lysogenic bacteriophage. However, with the caution suggested by the reviewer, we have changed the last sentence to: "This first observation invites us to pay more attention to the fecal-oral transmission route of the virus and suggests as a further possible reservoir of the virus also the bacteria of the human gut microbiome." Line 298-300 Overall, this is an interesting finding and corroborates the earlier report of the same author (F1000Res. 2021 May 11; 10:370) about the presence of the SARS-Cov2 like virus particles in bacterial culture. This report is an advancement as some evidence of the presence of the virus like particles have been shown using TEM, immune-fluorescence microscopy. The presence of SARS-Cov2 in feces has been documented before and as such this report does not add anything new to this however what is interesting is the replication potential of the virus in bacterial culture. The authors might want to provide more assays and evidence to showcase the "phage-like" activity of the SARS-Cov2 as they propose. Finally, please crosscheck the text. Lots of grammatical and contextual errors are found, some of which have been highlighted above.
The work was reviewed by a native speaker.