Microbiota in COVID-19 Patients for Future Therapeutic and Preventive Approaches

In December 2019, severe pneumonia cases were reported in relation to the Huanan Seafood
Wholesale Market in Wuhan, China. Four months and more than thousand deaths later, the
responsible pathogen of the largest and most critical global health emergency in the last 100
years is known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The
Coronavirus Disease 2019 (COVID-19) is characterized as a transmittable disease with a long
incubation period of between 7 and 14 days. In around 10% of cases a severe disease course is
observed. While predisposing factors such as older age, chronic arterial hypertension,
diabetes, as well as other comorbidities have been described, little is known about the
pathophysiological mechanism, which induce acute lung failure, coupled to the often-appearing
heart, kidney and vascular injury pathognomonic for the deadly course of this disease.

The alveolar exudative and interstitial inflammation impairing alveolo-capillary gas exchange
described in relation to SARS-CoV-2 complies with the definition of acute respiratory
distress syndrome (ARDS). The pathophysiological mechanics behind this injury to the lung
have been best described in bacterial sepsis induced ARDS, and are mainly induced by the
extravasation of neutrophil granulocytes from the capillary vasculature into the lung. One of
the main mediators of neutrophil extravasation is the degeneration of the endothelial surface
layer, namely the glycocalyx, induced by endotoxin-activated heparan sulfate. The thinning
and destructuration of the glycocalyx reveal hidden endothelial surface adhesion molecules
like VCAM-1 and ICAM-1. The thereby increased adhesion of Neutrophils coupled to the
inflammatory cytokine mediated dis-placement of VE-cadherin to the insides of the endothelial
cells, loosening the endothelial tight junctions, allows for an increased extravasation of
neutrophils and diffusion of protein-rich fluid into the interstitium. The thereby created
milieu induces neutrophil granulocytes activation and degranulation, and provokes the release
of toxic mediators that destroy the alveolar epithelium and produce further immunocytokines,
induce a cytokine storm and augment neutrophil recruitment. Finally the aggregation of the
inactivated pulmonary surfactant by the protein rich edema con-joined with the degenerated
type 1 alveolar epithelial cells and the hyaline membrane covered, de-nuded alveolar basement
membrane disrupt the gas exchange capacity of the alveolo-capilar membrane, impairing blood
oxygenation and decarboxylation.

In contrast to bacterial-induced ARDS, viral agents causing ARDS mainly reach the alveolar
epithelium through viral transport from the nasopharynx, from the upper to the lower
respiratory tract. In viral infections, the damage to the lung is primarily caused by a
direct viral invasion of type 1 and 2 pneumocytes. This causes the accumulation of
protein-rich edema in a two-fold fashion by disabling the ENaC channels, mainly responsible
for the decongestion of the alveolar room through osmotic gradient creation, and by breaching
the physio-chemical barrier established by the pneumocytes. Albeit the effect of alveolar
barrier disruption by means of vascular endothelial dysfunction being reduced in contrast to
bacterial sepsis, with the ARDS advancing, both the endothelium and epithelium secrete
chemotactors to attract macrophages and neutrophils to the inflamed lung, which in analogy to
bacterial ARDS induce a secondary damage to the lung as already described. However point to
an exaggerated initial capillary leak as compared to other forms of ARDS, which strongly
suggests endothelial dysfunction as a primary mechanism.

Endothelial cell damage may be assessed using both glycocalyx degradation products such as
syndecan-1, heparan sulfate, and VE-cadherins and direct visualization of red blood cell flow
properties within the capillaries via handheld vital microscopy (HVM) employing the dark
field microscopy technique. Recent advances in the investiagtors group have enabled the
accurate differentiation of modes of microcirculatory failure by quantification of the
microcirculatory diffusion and convection capacity. From a global perspective, the alveolar
leakage has previously been quantified using transpulmonary thermodilution. These tools
provide the optimal prerequisites to specifically detect (glycocalyx degradation products in
the alveolar lavage fluid) and quantify (transpulmonary thermodilution) endothelial cell
damage in the lung during ARDS caused by SARS-CoV-2 infection. Further, approximately 20% of
critically ill patients suffering from ARDS due to SARS-CoV-2 infection have been described
in preliminary reports to develop severe systemic inflammatory response and circulatory
shock. In these patients, the relation between alveolar endothelial cell damage and systemic
endothelial cell damage is of central interest and may be assessed by concomitant sublingual
HVM measurement.

Following the viral and inflammatory mediated lung damage as the initial trigger for ARDS,
the further deterioration of the lung function may mainly depend upon superinfection.
Bacterial and fungal superinfection has been described as mainly responsible for morbidity
and mortality in mid- to late-phase viral ARDS during similar pandemics, such as the Spanish
Flu in 1918. In the current outbreak in China, presence of bacterial and fungal
superinfections was found in 10 to 30%. Superinfection seemed to represent a major risk
factor for mortality in the COVID-19 patients. Albeit being unclear if detection of bacterial
and fungal superinfection has a clinical and therapeutic relevance, several authors advocate
empirical antibiotic treatment targeting mainly S. aureus and S. pneumonia.

Not only is the stratification of risk associated with bacterial and fungal superinfections
relevant to the assessment of severity of disease progression, but the question remains to be
assessed to which degree the presence of nosocomial pathogen transmission during pandemics
exists and is responsible for morbidity and mortality in hospitalized patients. Infection
prevention measures, such as standard and isolation precautions, are intended to prevent the
nosocomial transmission of pathogens in healthcare settings. These measures include hand
hygiene, the use of personal protective equipment (PPE) (e.g. isolation gowns, gloves, masks,
respirators, eye protection), and environmental measures - such as the cleaning and
disinfecting of surfaces and medical equipment and instruments. Yet, mobile devices, such as
private and professional mo-bile phones, have largely been unaddressed by such guidelines.
These devices may play an important role in indirect contact transmission. That is, when a
contaminated intermediate object is involved in the transfer of an infectious pathogen
between two individuals.

Current evidence, although limited, has shown an increased viability of SARS-CoV-2 on plastic
and stainless steel, materials frequently employed on mobile devices and protective covers,
of up to 72 hours. Albeit incorrect hand hygiene, lack of personal protective equipment and
other hygienic routine mistakes are the most obvious vectors of nosocomial pathogens, mobile
phones and other handheld devices have been found to be insufficiently disinfected, allowing
pathogen colonization, of which S. aureus is the most frequent. This is aggravated by the
fact that healthcare providers touched mobile objects, including mobile phones, on average
once every 6.9 seconds and touched their own body or face every 40 seconds. This was
accompanied by an extremely low adherence to hand hygiene protocols prior to colonization and
infection events. These findings underline the potential of mobile devices, to act as vectors
of transmission of pathogens to both patients and healthcare providers.

In combating viral infections, the innate immune defense recognizes viral infection mainly
through binding of pathogen associated molecular patterns (PAMPs) to pathogen-recognition
receptors (PRRs). The expression cascade induced by these receptor bindings stimulates the
Interferon (IFN) Type I and III pathway. The recruitment of the inflammatory
monocyte-macrophage-neutrophil axis by the IFN I pathway is essential in the fight against
most viral infections. Neverthe-less, viruses such as the Influenza A virus are capable of
evading the IFN type I and III inception, by moving their replication machinery into the cell
nucleus thus evading the cytoplasmically located PRRs or inactivating interferon mRNAs
through proteins. The IFN I pathway can act two-fold in these viruses; on one side it may
mediate initial action against the virus reducing maximal reached titers, on the other hand,
under certain circumstances it can nevertheless pathologically overex-press inducing a
cytokine storm and increasing damage to the lung parenchyma.

SARS-CoV and SARS-CoV-2 are positive-strand RNA viruses, which penetrate the cellular wall of
the infected cells by ACE-2 receptor mediated endocytosis. A onset of the IFN I pathway
expression may be associated with a stronger IFN II pathway activation and pro-inflammatory
cytokine storm induction and consequently a more severe dysregulation of the inflammatory
monocyte-macrophage system and the degree of lung immunopathological damage. The
pathophysiological mechanics of this new SARS-CoV-2 induced ARDS and the systemic
dysregulations it induces can only be speculated based on its predecessor SARS-CoV, and
insights elucidating the unusual clinical presentation of the novel disease are desperately
needed to derive an optimal treatment approach.

In summary, the mechanisms of disease severity in critically ill SARS-CoV-2 ARDS are
manifold, ranging from a dysregulated inflammatory response, over the presence of bacterial
and fungal superinfections to the importance of nosocomial transmission as a key targetable
element in day-to-day patient care. Only a global assessment of all facets possibly
responsible for the severity of dis-ease progression in COVID-19 patients, can aim to
elucidate the underlying concoction.

Project Objectives The overarching aim of this research is to gain the pathophysiological
understanding to improve morbidity and mortality in ARDS due to SARS-CoV-2 infection. This
research thus focuses on inflammation, microcirculatory dysfunction and superinfection,
aiming to elucidate risk factors for the development of severe ARDS of SARS-CoV-2 infected
patients and contribute to the rationale for therapeutic strategies.

The individual working hypotheses are:

1. The primary damage to the lung in SARS-CoV-2 ARDS is thought to be mediated by an
exaggerated pro-inflammatory response causing primary endothelial dysfunction, and
subsequently acting two-fold on the degradation of the lung parenchyma, through the
primary cytokine response, and through recruitment of the
inflammatory-monocyte-lymphocyte-neutrophil axis. The pronounced inflammation and
primary damage to the lung disrupt the pulmonary microbiome, leading secondarily to
pulmonary superinfections.

2. Pulmonary bacterial superinfections (i.e., S. pneumoniae, S. aureus, P. aeruginosa and
others) are a significant cause of morbidity and mortality in COVID-19 patients.
Pathogen colonization in the upper airways is a prerequisite and the main risk factor
for lower respiratory tract infections. To establish colonization, pathogens have to
interact with the local microbiota (a.k.a. microbiome) and certain microbiome profiles
will be more resistant to pathogen invasion.

3. Handheld devices used in clinical routine are a carrier of both, SARS-CoV-2, as well as
bacteria causing nosocomial pneumonia, making them a potential reservoir for indirect
contact transmission. A targeted infection prevention intervention can reduce this
contamination and thereby reduce the risk of nosocomial transmission.