Table 3.

Studies investigating the association between airway microbiota and COVID-19 infection

Study	Study type	Country	Sample size	COVID-19 Patients’ median age in yearsa	Study objective	Addressing co-relationship between microbiota and serum inflammatory markers	Antimicrobial and/or probiotic administration in participants	Sample collection and evaluation methods	Key findings
De Maio et al. [36]	Cohort study	Italy	Total: 40 (18 mild COVID-19 patients, 22 healthy individuals)	Not reported	To compare the nasopharyngeal microbiota composition among COVID-19 and non-COVID-19 patients	No	No report of antibiotic/probiotic use in the study	Samples: Nasopharyngeal swabs collected after COVID-19 infection No report of antibiotic/probiotic use before or after swab collection in the study Assessment method: 16 s rRNA sequencing	No significant differences in microbiota richness, diversity and composition between mild COVID-19 and control groups
Rueca et al. [37]	Cohort study	Italy	Total: 39 (10 COVID-19 ICU patients, 11 mild to moderate COVID-19 patients, 8 other coronaviruses patients, 10 healthy individuals	50	To investigate the difference of nasopharyngeal microbiota composition among COVID-19 patients, other coronavirus patients and healthy individuals	No	No report of antibiotics/probiotic use in the study	Samples: Nasopharyngeal swabs obtained after COVID-19 infection No report of antibiotics/probiotic use before or after sample collection in the study Assessment method: 16 s rRNA sequencing	Altered nasopharyngeal microbiota richness among COVID-19 patients, particularly the ICU patients Absence of Deinococcus Thermus, Alicyclobacillaceae, Chromobacteriaceae, Deinococcacaee, Hydrogenophilaceae, Thermoanaerobacteraceae, Sporomusaceae and Thermoanaerobacterales FamilyIII. Incertae Sedis, Johnsonella, Tepidiphilus, Thermoanaerobacter, Ther- moanaerobacterium, Thermosinus and Variovorax in COVID-19 patients and other coronavirus patients Complete depletion of BPB Bifidobacterium and Clostridium in COVID-19 ICU patients Significant decrease in Candidatus Saccharibacteria in COVID-19 ICU patients and other coronavirus patients compared to healthy individuals Opportunistic pathogens Salmonella, Scardovia, Serratia and Pseudomonadaceae were only found in COVID-19 ICU patients Bulleidia, Halanaerobium, Streptobacillus, Epsilonproteobacteria Moraxellaceae, Mycoplasmataceae and Tenericutes were only found in paucisymptomatic COVID-19 patients Pectobacteriaceae were found exclusively to SARS-CoV-2 ICU patients
Shen et al. [43]	Cohort study	China	Total: 53 (8 COVID-19 patients, 25 community-acquired pneumonia (CAP) patients, 20 healthy individuals)	Not reported	To examine the mutation rate of COVID-19 and also the variances of nasopharyngeal microbiota between COVID-19 and community-acquired pneumonia (CAP) patients	No	Pneumonia patients included in study received antibiotic Study did not describe the period of antibiotic use	Samples: Bronchoalveolar lavage fluid through bronchoscope Sample collection was performed after COVID-19 infection Study did not describe if antibiotics were used before or after the sample collection Assessment method: Metatranscriptome sequencing	No significant differences on the microbiota profile of COVID-19 and CAP patients 25% of COVID-19 sample had more than 5% variation(SARS-CoV-2 mutation rate comparable to Ebola virus)
Nardelli et al. [39]	Cohort study	Italy	Total: 38 (18 COVID-19 patients, 8 recovered COVID19 patients, 12 healthy individuals)	Not reported	To examine any differences in nasopharyngeal microbiota composition among recovered COVID-19 patients, current COVID-19 patients and healthy individuals	No	No report of antibiotics/probiotic use in the study	Samples: Nasopharyngeal swabs were obtained after COVID-19 infection No report of antibiotics/probiotic use before or after sample collection in the study Assessment method: 16S rRNA sequencing	Microbiome significantly different in COVID-19 group compared to the control group Difference in microbiota composition remained after patients’ recovery Fusobacterium periodonticum may increase the susceptibility to COVID-19 infection Significant increase of Firmicutes, Bacteroidetes, Actinobacteria in COVID-19 group compared to control group Significant decrease of Proteobacteria, Fusobacteria, Leptotrichia and Haemophil us compared to A further reduction of Fusobacterium was reported in more severe patients compared to less severe COVID-19 patients
Ventero et al. [40]	Cohort study	Italy	Total: 74 (19 mild COVID-19 patients without hospitalisation, 18 severe COVID-19 patients with hospitalisation, 19 critical COVID19 patents admitted to intensive care units, 18 COVID-19 negative individuals with comorbidities)	66	Relationship of nasopharyngeal microbiota composition and COVID-19 severity	No	No report of antibiotics/probiotic use in the study	Samples: Nasopharyngeal swabs collected after COVID-19 infection No report on whether the antibiotics used were or after the sample collection Assessment method: 16 s rDNA sequencing	Marked alteration of nasopharyngeal microbiota composition between COVID-19 and control groups Decreased Network complexity of the microbiota was associated with with more severe disease Significant increase of Firmicutes, Bacteroidota, Proteobacteria, Actinobacteria in covid-19 patients compared to non-COVID-19 patients Significant increase of opportunistic pathogens Streptococcus, Prevotella, Veillonella, Haemophilus, Moraxella and Leptotrichia in covid-19 patients compared to non-COVID-19 patients Higher abundance of Prevotella was found in more severe COVID-19 patients compared to less severe COVID-19 patients
Rosas-Salazar et al. [38]	Cohort study	USA	Total: 59 (38 mild-moderate symptomatic COVID-19 patients, 21 uninfected healthy control)	30	To examine the difference in composition of airway microbiome between COVID-19 and non-COVID-19 subjects	No	Participants included in study did not receive any antibiotics for the previous 2 weeks or use any current intranasal medications No report of antibiotics/probiotic use in the study	Samples: Nasopharyngeal swabs collected after COVID-19 infection Study did not describe whether antibiotics were used after sample collection Assessment method: 16 s rRNA sequencing	Higher species index of upper respiratory tract microbiota in COVID-19 group compared to healthy control group Marked alteration of airway microbiota composition in COVID-19 patients compared to healthy control group Viral load proportional to the level of alteration of airway micromiome Increased abundance of Corynebacterium, Lawsonella,Staphylococcus, Dolosigranulum and Peptoniphulus in COVID-19 patients compared to healthy control group Increased abundance of Corynebacterium_1,, Moraxella, Dolosigranulum Staphylococcus, and Neisseria in non-COVID-19 subjects compared to COVID-19 subjects More abundance of Neisseriacea, Anaerococcus, Peptoniphulus, Campylobacter, and Enterococcus in COVID-19 patients wih higher viral load compared to healthy control group More abundance of Corynebacterium_1, Staphylococcus, Granilucatella, Neisseria, and Prevotella in COVID-19 patients with lower viral load
Miao et al. [41]	Cohort study	China	Total: 397 (229 mild covid-19 patients, 78 severe covid-10 patients, 16 critical COVID-19 patients, 20 intubated non-COVID19 patients, 31 non-intubated non-COVID viral pneumonia patients, 23 non-intubated healthy subjects)	70.5	To investigate co-infection rate and rate of antimicrobial usage among COVID-19 patients across disease severity To examine any differences in airway microbiota composition between critical COVID-19 patients and other non-COVID-19 patients	No	48 mild COVID-19 cases received antibiotics, none received carbapenems 60 severe COVID-19 cases received antibiotics and 3 received carbapenems 16 of critically severe COVID-19 cases received antibiotics and 13 received carbapenems Study did not describe whether antibiotics were given before, after or during COVID19 infection	Samples: Nasopharyngeal swabs (Bronchoalveolar fluid lavage, Endotracheal aspiration) Sample collection performed after COVID-19 infection Study did not describe whether the antibiotics used were used before or after the sample collection Assessment method: Metagenomic Next-generation Sequencing	Significantly higher co-infection rate among critical COVID-19 patients (81.3%) compared to severe patients (5.1%) and mild patients (0%) Klebsiella, Enterococcus, Coagulase-negative Staphylcocci, S. wiggsiad and M. hominis were the most common bacterial causes of co-infection in COVID-19 patients Candida, Aspergillus and Cryptococcus were the most common fungal causes of co-infection in COVID-19 patients Cytomegalovirus, Herpes Simplex Virus, Epstein-Barr Virus, Torque Teno Virus, Human Parvovirus B19 and JC Polyomavirus were the most common viral causes of co-infection in COVID-19 patients Anti-microbials commonly used in COVID-19 patients (21% mild patients, 76.9% severe patients, 100% critical patients) Alteration of airway microbiome profile in critical COVID-19 patients was likely due to intubation, rather than COVID-19 infection
Braun et al. [44]	Case control study	Israeli	Total: 33 (21 COVID-19 positive subjects, 12 COVID-19 negative subjects)	52	To examine any differences in airway microbiota composition in COVID-19 positive and negative samples	No	No report of antibiotics/probiotic use in the study	Samples: Nasopharyngeal swabs were obtained after COVID-19 infection Study did not report antibiotics/probiotic use before or after swab collection Assessment method: 16 s rRNA sequencing	COVID-19 did not insert any significant effect on the composition of airway microbiota No significant difference between COVID-19 positive and negative groups
Zhang et al. [42]	Cohort study	China	Total: 187 (62 COVID-19 patients, 125 non-COVID pneumonia patients)	Not reported	To investigate changes of diversity of airway microbiome among COVID-19 patients Gene markers to better diagnose the disease	Yes	No report of antibiotics/probiotic use in the study	Samples: Nasopharyngeal swabs from sputum samples Swabs collected after COVID-19 infection Study did not report antibiotic/probiotic usage before or after swab collection Assessment method: RT-PCR and Metatranscriptomic NGS Sequencing	Airway microbiome in COVID-19 samples were less diversified Certain microbiota were associated with CRP concentration 47.4% of COVID-19 samples revealed an increase of presence in opportunistic pathogens compared to 52% of non-COVID-19 samples 36 differentially expressed genes related to immune pathway such as cytokine signalling were found in COVID-19 samples, suggesting a possible diagnostic marker for COVID-19 Increased abundance of Human influenza virus, Respiratory syncytial viruses, Human alphaherpesvirus 1 and Candida albicans in COVID-19 patients compared to non-COVID-19 patients
Mostafa et al. [45]	Cohort study	USA	Total: 50 (40 COVID-19 patients, 10 patients suspected with COVID-19 infection but with negative results)	50.5	To examine the accuracy of metagenomic next-generation sequencing (mNGS) on COVID-19 diagnosis, the Coinfection in COVID-19 patients and any Changes in the composition of the airway microbiome	No	No report of antibiotic/probiotic use in the study	Samples: Nasopharyngeal swabs collected after infection or suspected with COVID-19 No report of antibiotic/probiotic usage before or after swab collection in the study Assessment method: metagenomic sequencing	mNGS achieved 77.5% accuracy compared to traditional method RT-PCR 12.5% of COVID-19 positive samples contained other opportunistic pathogens Significant decrease in their diversity of the airway microbiota composition in COVID-19 patients, especially in more severe infection compared to negative COVID19 patients High abundance of opportunistic pathogens, including Haemophilus influenzae, Moraxella catarrhalis, human metapneumovirus and human alphaherpesvius in COVID-19 positive samples compared to the negative samples
Merenstein et al. [46]	Cohort study	USA	Total: 113 (83 hospitalised COVID-19 patients <  30 healthy control)	64	To examine any alteration in the composition of airway microbiota in COVID-19 patents and its association with disease severity	Yes	72 COVID- 19 subjects received antibacterial agents 20 COVID- 19 subjects received antifungals agents 18 COVID-19 subjects received antiviral agents 13 Non-COVID-19 subjects received antibacterial Study did not mention the timing of antimicrobial agents used	Samples: Nasopharyngeal swabs from Endotracheal aspiration- Oropharyngeal sampling Samples obtained after COVID-19 infection Study did not report antibiotic/probiotic usage before or after swab collection Assessment method: 16S rRNA gene sequencing	The airway microbiome communities of COVID-19 were markedly different from that of the healthy control group The diversity of the microbiome was significant decreased in COVID-19 cases, especially in more severe patients compared to healthy control Level of dysbiosis was associated with COVID-19 severity The microbiome composition as associated with lymphocyte to neutrophil ratio and a specific peripheral blood mononuclear cell profile Unclear whether the systemic immune response was directed by the airway microbiota or other factors such as disease severity Lower abundance of Proteobacteria, Actinobacteria, Haemophilus, Actinomyces and Nisseria in severe COVID-19 samples compared to samples from healthy individuals Higher presence of Bacteroidtes, Anelloviridae, Redondoviridaee in COVID-19 samples compared to samples from healthy individuals Significant increase of opportunistic pathogens including Staphylococcus, Enterococcus, Stenotrophomonas, Enterobacteriaceae and Enterobacterales in COVID-19 patients compared to healthy control Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease
	Cohort studies = 10 Case control study = 1	Studies conducted in Italy = 4 Studies conducted in China = 3 Studies conducted in USA = 3 Studies conducted in Israel = 1	Mean = 98.45 SEM = 31.31	Mean = 54.71 SEM = 4.78	Airway microbiota composition during COVID-19 = 10 Coinfection in COVID-19 = 2 Association between airway microbiota and COVID-19 severity = 2 Airway microbiota composition and susceptibility of COVID-19 = 1	No = 9 Yes = 2	Studies in which antibiotic/probiotic use was not reported = 7 Studies in which antibiotic/probiotic use were included = 3 Studies in which antibiotic/probiotic use by partitcipants was excluded = 1	Studies which used samples from Nasopharyngeal swabs = 10 Studies which used samples from Endotracheal aspiration = 2 Studies which used samples from Bronchoalveolar fluid lavage = 2 Studies which used samples from Sputum samples = 1 Studies which used samples from Swabs are collected after COVID-19 infection = 11 Studies with no reports of antibiotics/probiotic use before or after swab collection in the study = 11 Study with no report of sample collection after treatment initiation = 4	Studies that reported increase in opportunistic pathogens = 6 Studies that reported decrease in beneficial bacteria = 2 Studies that reported no significant difference in microbiological composition = 3

^aIn cases of multiple groups of COVID-19 patients, the median age of the largest group is used in the analysis