Relationship between the nasopharyngeal microbiota and the severity of COVID-19


The microbiota in the nose and upper throat likely contains biomarkers for assessing how sick an individual infected with SARS-CoV-2 may get and for developing new treatment strategies to improve their outcome, researchers say.

This nasopharyngeal microbiota is generally considered a frontline protection against viruses, bacteria and other pathogens that enter these natural passageways, says Dr. Sadanand Fulzele, geriatric researcher in the Department of Medicine at the Medical College of Georgia at Augusta University.

Distinct patterns emerged when the researchers examined the microbiota of 27 individuals age 49 to 78 who were negative for the virus, 30 who were positive but had no symptoms, and 27 who were positive with moderate symptoms that did not require hospitalization, they report in the journal Diagnostics.

“Millions of people get infected and relatively few of them become symptomatic. This might be one of the reasons,” says Dr. Ravindra Kolhe, director of MCG’s Georgia Esoteric and Molecular Laboratory, or GEM Lab. which has performed more than 100,000 COVID tests.

The most significant changes were in those who were symptomatic, including about half those patients not having a sufficient amount of microbiota to even sequence, says corresponding author Fulzele.

They were surprised to find these “low reads” of bacteria in the nasopharyngeal cavity of symptomatic individuals versus only two and four individuals in the negative and positive with no symptoms groups, respectively. The vast majority of the positive individuals with no symptoms still had sufficient microbiota, notes first author Kolhe.

“We don’t know which came first, the disease or the wipeout of the microbiota,” Fulzele says. Runny noses and sneezing might account for the loss, an already significantly lower number of bacterial inhabitants might have increased the individuals’ risk for developing these kinds of symptoms, or the virus may have changed the landscape, says Fulzele, who suspects it’s the latter.

Based on experience with microbiota in the gastrointestinal tract, Kolhe thinks the different microbiota content and size is another good bet and they both would like a definitive answer. “We don’t have sufficient data at this moment,” Kolhe says.

They found differences in the type of bacteria as well, although the researchers note that the function of some of the bacteria they found are not well understood.

As the virus’ name and nearly two years of experience with it indicate, a major method for transmitting severe acute respiratory syndrome coronavirus 2, or SARS-Cov-2, is when someone coughs, sneezes or even talks, and droplets called aerosols carrying the virus move through the air and into another person’s nose or mouth.

Those age 65 and older and/or with underlying health conditions like hypertension and diabetes, are considered at increased risk for hospitalization and death from the infection, so they decided to look at the microbiota in the upper part of the respiratory system called the nasopharynx of older individuals.

The moist, mucus-producing lining of this area works like a natural barrier to invaders and there also is a significant complement of immune cells present, Fulzele says, and their response to respiratory viruses is key.

The area also is abundant with ACE-2 receptors, to which the spiky virus binds, and Kolhe says it’s a major landing spot for this virus.

Their new findings indicate that the altered microbiota in the symptomatic patients impacted their immune response to the virus, Kolhe and Fulzele say.

The symptomatic individuals had significantly higher levels of two bacterial species, including Cutibacterium, generally found on the skin and associated with acne but also with heart infection and shoulder infections following surgery. Conversely there was a significantly lower presence of a handful of other, not well-studied bacterium.

The microbiota of both infected groups, symptomatic and asymptomatic, had high levels of bacterium like Cyanobacteria, also called blue green algae, that can be found in contaminated water but is a usual inhabitant of the microbiome in humans which appears to have a role in regulating the immune response.

These bacterium typically enter the body through mucosal surfaces, like those in the nose, and are known to cause pneumonia and liver damage. Those who were symptomatic had twice as much of this bacterium as their asymptomatic counterparts.

Fulzele notes that between the asymptomatic and symptomatic there was no significant change in microbiota diversity—just those big differences in volume—but they did see a lot of individual bacterium moving up and down in numbers.

For example, their graph of the number of another water-loving bacterium Amylibacter, looked like stair steps as it moved from negative to positive with symptoms individuals, while there was a downward trend in a handful of other bacterium.

While the relationship between the nasopharyngeal microbiota and the severity of COVID-19 remains unknown, their study indicates a “strong association” between the nasal microbiota, SARS-CoV-2 infection and severity, they write.

Their analysis was done before the current virus variants began to surface, but the researchers say the differences in the microbiota likely will hold for these as well and they have already begun that analysis.

Larger studies are needed to ensure that the clear patterns they found hold, the researchers say. They are putting together a grant application that will enable a larger study and looking for other testing sites who want to be partners. Using the same nasopharyngeal swab used for many COVID tests would enable a microbiota analysis to be done at the same time as testing, they say.

They note the striking contrast that has emerged over nearly two years of experience with the virus, with the majority of those infected being asymptomatic or experiencing mild symptoms like they would with a cold, while others get severe viral pneumonia, require hospitalization and die.

A handful of recent studies have now been published suggesting that the bacterial composition of the nasal canal can have a “drastic” influence on the development of respiratory infections and the severity of symptoms, they write.

Some studies have indicated that the nasal microbiota can influence the viral load, immune response and symptoms of a rhinovirus infection, which is responsible for somewhere between 10-40% of common colds.

A myriad of other conditions like inflammatory bowel syndrome, peptic ulcers and viral diseases have been linked to significant changes in the microbiota of the gut, nasal and oral cavity, they write.

Diversity of bacterium in the microbiota is generally a good thing, and it’s something that naturally decreases with age, says Fulzele, and also can be harmed by habits like smoking and improved by those like eating a diverse diet.

Comparing the nasopharyngeal microbiomes of 79 SARS-CoV-2 positive samples, representing a range of COVID-19 severity, and 20 negative samples, revealed differences in diversity, frequency, and abundance of several species with respect to COVID-19 status.

While alpha diversity did not differ according to COVID-19 status, it was different with respect to age and smoking status. It should be noted that both age and smoking status have previously been shown to influence the gut, skin, and upper respiratory tract microbiome [21,22].

Beta diversity of the microbiome was significantly different for those who had COVID-19, used breathing assistive devices, had an inpatient hospital stay, and, sadly, for those who died. These factors often showed high overlap; breathing assistive devices were used only for inpatients suffering COVID-19, although not all who had COVID-19 needed breathing assistance or required an inpatient hospital stay. One patient who died was COVID-19 negative, but that the death was linked to other categories (see Supplemental Table S1 for specific patient details).

It is recognized that race is a social construct and not a biological factor, but race is shown repeatedly to be associated with health outcomes. Surprisingly, we observed racial differences in beta-diversity of the NP space, but with the caveat that the dispersion was significantly different between racial groups, suggesting that the differences in diversity can be driven by the wide variance rather than a biological factor. Research on racial differences with respect to the microbiome is a topic of high interest currently.

Investigators have reported that oral microbial richness and composition were different among African- and European-Americans in a study of more than 1500 individuals [23]. Importantly, this difference was noted for self-reported race as well as percent of those with genetically determined African ancestry.

The Healthy Life in an Urban Setting (HELIUS) study included 2084 participants and demonstrated ethnic differences in the diversity and abundance of species comprising the gut microbiome, despite the fact that all subjects were residents of Amsterdam, Netherlands [24]. A larger sample size will be required to determine whether actual racial differences exist, but this preliminary finding emphasizes that race should be considered in microbiome studies.

COVID-19 positivity was associated with increased representation of Serratia. It is important to note that Serratia marcescens is a recognized causative agent of human diseases, including pneumonia. Other investigators have noted the paradoxical risk of S. marcescens infection in intensive care units during the current pandemic [25]. Despite taking increased sanitation measures and great care in PPE donning and doffing, highly resistant S. marcescens infections occurred in five ICU patients in Paris, France, likely because of decreased hand hygiene related, ironically, to increased sterile glove use. [25]

Kocuria spp. were detected only in the COVID-19 positive patients and, although not at a significant level, it is curious that none of the COVID-19 negative samples contained this genus since it is ubiquitous in its occurrence on human skin and mucus membranes. Kocuria species have been identified as a causative agent in hospital acquired infections [26] but, in this case, Kocuria was present only in a subset of individuals taking antibiotics, a majority of whom (16/17) were not admitted to hospital.

This association was not determined to be significant, hence a larger study is needed to confirm the connection, if any.
Samples from patients who required breathing assistance showed a marked difference in the PCoA compared to those who did not and revealed an association with increased abundance of Serratia, Streptococcus, Enterobacter, Veillonella, Prevotella and Rothia.

On the other hand, while a subset of COVID-19+ patients had Finegoldia, an opportunistic human pathogen, and Peptoniphilus, typically associated with the gut and vaginal microbiota, these genera were absent in COVID-19+ patients requiring breathing assistance. Breathing assistive devices were employed only for inpatients, although not all inpatients required breathing devices.

Other investigators have noted ventilator-associated lung dysbiosis in COVID-19 patients with significantly higher rates of co-infection versus non-COVID-19 patients [27]. In patients with severe COVID-19 (n = 38 positive), that invasive mechanical ventilation greatly increased the risk of secondary infection, particularly bacterial infections in the respiratory system, bloodstream, and urinary tract [28].

The interplay between SARS-CoV-2 infection, breathing treatment, hospital stay, and respiratory microbiomes is complex, and a detailed timeline and/or longitudinal sampling of patients is required to identify causal relationships. Nonetheless, our results indicate that these factors should be considered when to evaluating microbiome structure and dynamics over time.

Reduction in Fusobacterium periodonticum associated with SARS-CoV-2 was reported in a study of Italian patients (n = 18 positive, 12 negative) [4], but was not observed in our study, nor did we observe significant difference in alpha diversity and COVID-19 status.

Propionibacteriaceae, or Corynebacteriam accolens, as was reported for a study of 50 patients (n = 40 positive, 10 negative) in Baltimore [3]. Another study found no differences in the nasopharyngeal microbiota of patients who had mild COVID-19 (n = 9 positive, 10 n

egative) [2]. Analysis of patient samples in Nashville (n = 38 mild to moderate positive, 21 negative) revealed the complexity of interaction between viruses and bacteria in the upper respiratory tract, as differences in the airway microbiome were reported to be dependent on the SARS-CoV-2 viral load [16].

Similarly, in the lower airway, samples obtained through bronchial lavage (n = 142 positives) were enriched with oral bacteria in COVID-19 patients who had worse clinical outcomes, but mortality was better predicted by viral load and host immune response [17].

While microbial differences were present, SARS-CoV-2 and host factors were the most important in disease severity in both studies. Finally, Clostridium botulinum, Bacillus cereus and Halomonas spp. were found in almost all samples in an Indian study focused on more severe COVID-19 [5], which was clearly incompatible with our findings.

This study did not include COVID-19 negative controls, which may have helped to distinguish between species commonly found in the NP space for the region in which they were collected versus those that are enhanced or lost during SARS-CoV-2 infection. In summary, viewed across the many studies to date, including the one reported here, differences in patient populations, geography, and climate, or even quantity or genomic diversity of the SARS-CoV-2 virus itself all play some role in the NP microbiome.

Reliance on retrospective medical chart review is recognized to be a limitation of the study reported here. At the time of data collection, direct patient contact or interviewing was difficult or impossible due to limitations of both PPE supply and staffing. It is well known that antibiotics dramatically change the gut microbiome [29].

Thus, future research will need to investigate specific medication used, with respect to the NP microbiome. Additionally, antibiotic use can cause changes in the gut biome for a few weeks or up to six months [30] and antibiotics taken by patients for up to six months earlier may also have had a lasting effect on the NP microbiome, which this study was not designed or powered to detect.

reference link :

More information: Ravindra Kolhe et al, Alteration in Nasopharyngeal Microbiota Profile in Aged Patients with COVID-19, Diagnostics (2021). DOI: 10.3390/diagnostics11091622


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