Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infection during SARS-CoV-2 Infection

  1. Amanda P. Smith
  2. Evan P. Williams
  3. Taylor R. Plunkett
  4. Muneeswaran Selvaraj
  5. Lindey C. Lane
  6. Lillian Zalduondo
  7. Yi Xue
  8. Peter Vogel
  9. Rudragouda Channappanavar
  10. Colleen B. Jonsson
  11. Amber M. Smith

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Abstract

Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate SARS-CoV-2 infection increased the risk of pneumococcal coinfection in a time-dependent, but sexindependent, manner in the transgenic K18-hACE mouse model of COVID-19. Bacterial coinfection was not established at 3 d post-virus, but increased lethality was observed when the bacteria was initiated at 5 or 7 d post-virus infection (pvi). Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.

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    SciScore for 10.1101/2022.02.28.482305: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIACUC: All experimental procedures were performed under protocol 20-0132 approved by the Animal Care and Use Committee at University of Tennessee Health Science Center (UTHSC) under relevant institutional and American Veterinary Medical Association (AVMA) guidelines and were performed in a biosafety level 3 facility that is accredited by the American Association for Laboratory Animal Science (AALAS).
    Sex as a biological variableMice: Adult (10-13 week old) male and female K18-hACE2 transgenic mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from Jackson Laboratories (Bar Harbor, ME).
    Randomizationnot detected.
    BlindingStained sections were counterstained with hematoxylin, dehydrated, and examined by a pathologist blinded to the experimental group assignments.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The followed anti-mouse antibody panels were used for cell subset analysis: CD45 (clone 30-F11, Pe-Cy7, Biolegend),
    anti-mouse
    suggested: None
    CD45
    suggested: None
    Pe-Cy7
    suggested: None
    For IHC, a primary monoclonal antibody against SARS-CoV-2 nucleoprotein (NP) (Sino Biological, Wayne, PA) was used at 1:1000 followed by a biotinylated anti-rabbit antibody (Vector Laboratories, Burlingame, CA) at 1:200, the Vectastain Elite ABC-HRP kit (Vector Laboratories, Burlingame, CA), and 3,3’-Diaminobenzidine (DAB) solution development.
    SARS-CoV-2 nucleoprotein ( NP
    suggested: None
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The viral infectious dose [plaque forming units (PFU)] was determined by plaque assay of serial dilutions on Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: Adult (10-13 week old) male and female K18-hACE2 transgenic mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from Jackson Laboratories (Bar Harbor, ME).
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Software and Algorithms
    SentencesResources
    The data were analyzed using FlowJo 10.7.2
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The areas of both the entire lung parenchyma (alveoli and bronchioles) and the virus-positive regions were outlined manually with areas determined using ImageScope software (Aperio Technologies, Inc.).
    ImageScope
    suggested: (ImageScope, RRID:SCR_014311)
    Linear values of lung and blood bacterial loads, viral loads, immune cells, cytokines/chemokines were compared using unpaired t-tests with Welch correction or Mann-Whitney test where appropriate (GraphPad Prism 9.2.0 and Rv4.0.3).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2022.02.28.482305v1 on bioRxiv