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.