key: cord-0330931-hhn2m8wz
authors: Ventero, Maria Paz; Moreno-Perez, Oscar; Molina-Pardines, Carmen; Paytuví-Gallart, Andreu; Boix, Vicente; Galan, Irene; González-delaAleja, Pilar; López-Pérez, Mario; Sánchez-Martínez, Rosario; Merino, Esperanza; Rodríguez, Juan Carlos
title: Nasopharyngeal Microbiota as an early severity biomarker in COVID-19 hospitalised patients: a retrospective cohort study in a Mediterranean area
date: 2021-09-28
journal: bioRxiv
DOI: 10.1101/2021.09.28.461924
sha: 26fa077cd59b04b718c689f45e33badf94535527
doc_id: 330931
cord_uid: hhn2m8wz

Background There is mounting evidence suggesting that the microbiome composition could be different in COVID-19 patients. However, the relationship between microbiota and COVID-19 severity progression is still being assessed. This study aimed to analyse the diversity and taxonomic composition of the nasopharyngeal microbiota, to determine its association with COVID-19 clinical outcome. Methods and Findings Samples came from a retrospective cohort of adult patients with COVID-19, hospitalised in a tertiary centre. To study the nasopharyngeal microbiota, we utilized 16S rRNA sequencing. Raw sequences were processed by QIIME2. The associations between the microbiota, invasive mechanical ventilation (IMV), and all-cause mortality were analysed by multiple logistic regression (OR; 95%CI), adjusted for age, gender, and comorbidity. 177 patients were included: median age 68.0 years, 57.6% males, 59.3% had a Charlson comorbidity index ≥3, and 89.2% with pneumonia. The microbiota α diversity indexes were lower in patients with a fatal outcome, and this association persisted after adjustment for the main confounders; whereas the β diversity analysis showed a significant clustering, grouping the patients with a fatal outcome. After multivariate adjustment, the presence of Selenomonas spp., Filifactor spp., Actinobacillus spp., or Chroococcidiopsis spp., was associated with a reduced risk of IMV (adjusted OR 0.06[95%CI 0.01–0.0.47], p = 0.007). Conclusions The microbiota diversity and taxonomic composition are related to COVID-19 severity. Higher diversity and the presence of certain genera in the nasopharyngeal microbiota seem to be early biomarkers of a favourable clinical evolution in hospitalised patients with moderate to severe SARS-CoV-2 infections.

Introduction 52 In this time of pandemic finding early prognostic markers of COVID-19 severity is of 53 utmost importance [1, 2] . It is known that poor outcomes related to COVID-19 are not 54 only a consequence of the viral infection, but are also related to an aberrant host 55 immune response, including the vast release of cytokines by the immune system, 56 leading to uncontrolled inflammation and multi-organ failure [3] . 57 Several risk or prognostic factors, such as genetic factors, comorbidities, age, sex, and Additionally, some studies have shown a relationship between the composition of the 71 gut and respiratory microbiota and disease severity [12] . This relationship appears to be 72 mainly based on the capacity of the microbiota to modulate the immune response 73 [13, 14] , through modification of the gut-lung axis [12, 15, 16] , and to alter the expression of angiotensin-converting enzyme 2 (ACE2) receptors, which are used by SARS-CoV-2 to 75 enter host cells [17, 18] . 76 The available evidence suggests a potential role of microbiota in susceptibility to CoV-2 infection and COVID-19 severity, but longitudinal studies evaluating the 78 microbiota as a prognostic factor for severity of disease progression are lacking. The 79 data regarding the association between nasopharyngeal microbiota features and 80 disease severity are scarce, and limited in terms of showing a decrease in α diversity or 81 identifying specific genera with relevance to critical illness [19, 20] . Since the sampling of 82 this location is very accessible, with the nasopharyngeal aspirate swab diagnostic 83 confirmation procedure able to obtain this information, it should be a priority to address 84 the relationship between nasopharyngeal microbiota and COVID-19 outcomes. 85 This study aimed to analyse the nasopharyngeal microbiota from hospitalised COVID-19 86 patients, to determine the relationship between the microbiota and SARS-CoV-2 87 infection clinical outcomes and to identify features or genera that could be used as 88 severity prognostic markers. (Figs 1A, 1B, and 1C) . 183 The protective effect of a greater microbiota diversity persisted for the Shannon grouping together the fatal outcome patients (Fig 1D) . In the case of IMV, neither the α 188 diversity indexes nor β diversity analyses showed any significant differences. detected in non-IMV patients (Fig 2A) . (Fig 2B) . 211 Differently represented genera and outcomes 212 This study was performed to identify differential genera between the subpopulations 213 with and without specific outcomes. We found that Selenomonas spp. ( (Fig 2C) . after adjustment for the main confounders in the multivariate model (Fig 3) . (Fig 2D) . The presence of Actinobacillus spp., Citrobacter spp., 228 Craurococcus spp., or Moheibacter spp., was associated with a reduced risk of a fatal 229 outcome (OR 0.309[95%CI 0.10-0.93], p = 0.037). This association did not persist after 230 adjustment for the main confounders in the multivariate model (Fig 3) . results. Finally, the 16S ribosomal RNA amplicon sequencing approach to study the 315 microbiota could introduce bias in the obtained data because this method does not 316 allow the study of the whole microbiome, but only the genera amplified by PCR. 317 Nevertheless, it is the most common technique to study microbiota in clinical samples. 318 Moreover, the microbiota bioinformatics analysis has not been standardized yet, which 319 hampered comparison interpretations of our results. 320 In summary, the higher diversity found in patients without IMV or a fatal outcome, 

Microbiome Alterations and Secondary Bacterial Pneumonia

Which Came First, the Chicken or the Egg? Clin Infect Dis

The 374 human microbiome and COVID-19: A systematic review

Gut microbiota composition 377 reflects disease severity and dysfunctional immune responses in patients with 378 COVID-19

Lung microbiome and coronavirus disease 2019 (COVID-380 19): Possible link and implications

COVID-19 and the Gut Microbiome: More than a Gut 383

SARS-CoV-2 385 microbiome dysbiosis linked disorders and possible probiotics role. Biomedicine 386 and Pharmacotherapy

COVID-19 -Gastrointestinal 389 and gut microbiota-related aspects

Immunological co-ordination between gut and lungs 392 in SARS-CoV-2 infection

Variability in digestive and 395 respiratory tract Ace2 expression is associated with the microbiome

Metagenomic analysis reveals 398 oropharyngeal microbiota alterations in patients with COVID-19

With SARS-CoV-2 That Developed COVID-19. Front Microbiol

Using 405 QIIME to analyze 16S rRNA gene sequences from microbial communities. Curr 406 Protoc Microbiol

The SILVA 409 ribosomal RNA gene database project: Improved data processing and web-based 410 tools

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

Gut Microbiome Composition Is Associated with COVID-19 415 Disease Severity

Gut microbiota and Covid-19-possible link and implications

Alterations of the Gut Microbiota 420 in Patients With Coronavirus Disease 2019 or H1N1 Influenza

Alterations in Gut 423 Microbiota of Patients With COVID-19 During Time of Hospitalization

Gut microbiota, inflammation, 426 and molecular signatures of host response to infection

Gut microbiota in the 429 pathogenesis of inflammatory bowel disease

SARS-CoV-2 does 432 not have a strong effect on the nasopharyngeal microbial composition

Nasopharyngeal Microbiota Profiling of SARS-CoV-2 Infected Patients

Association between the 438 nasopharyngeal microbiome and metabolome in patients with COVID-19

Metagenomic next-generation sequencing of nasopharyngeal specimens 442 collected from confirmed and suspect covid-19 patients

Nasopharyngeal Microbiota in SARS-CoV-2 Positive and Negative Patients

Metagenomic comparison of tracheal aspirate and mini-bronchial alveolar lavage 449 for assessment of respiratory microbiota

The 452 Role of Bacterial and Fungal Human Respiratory Microbiota in COVID-19 Patients. 453 BioMed Research International. Hindawi Limited

The Human Microbiome and Its Impacts on Health

The lower 459 respiratory tract microbiome of critically ill patients with COVID-19