Massilia litorea sp. nov., Marinobacter salinisoli sp. nov. and Rhodobacter xanthinilyticus sp. nov., isolated from coastal environments.

Three bacterial strains, namely LPB0304T, LPB0319T and LPB0142T, were isolated from coastal environments. The 16S rRNA gene sequences of the three isolates were found to show the highest sequence similarities to Massilia litorea (98.44 %), Marinobacter salinisoli (97.55 %) and Rhodobacter lacus (97.60 %), respectively. The low (<98.7 %) sequence similarities and tree topologies implied the novelty of the three isolates, representing novel genomic species of the genus Massilia, Marinobacter and Rhodobacter. Numerous biochemical and physiological features also supported the distinctiveness of the isolates from previously known species. Based on the phenotypic and phylogenetic data presented in this study, three novel species are suggested with the following names: Massilia litorea sp. nov. (LPB0304T=KACC 21523T=ATCC TSD-216T), Marinobacter salinisoli sp. nov. (LPB0319T=KACC 21522T=ATCC TSD-218T) and Rhodobacter xanthinilyticus sp. nov. (LPB0142T=KACC 18892T=JCM 31567T).

Universal primers for rift valley fever virus whole-genome sequencing.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease causing acute hemorrhagic fever. Accurate identification of mutations and phylogenetic characterization of RVF virus (RVFV) require whole-genome analysis. Universal primers to amplify the entire RVFV genome from clinical samples with low copy numbers are currently unavailable. Thus, we aimed to develop universal primers applicable for all known RVFV strains. Based on the genome sequences available from public databases, we designed eight pairs of universal PCR primers covering the entire RVFV genome. To evaluate primer universality, four RVFV strains (ZH548, Kenya 56 (IB8), BIME-01, and Lunyo), encompassing viral phylogenetic diversity, were chosen. The nucleic acids of the test strains were chemically synthesized or extracted via cell culture. These RNAs were evaluated using the PCR primers, resulting in successful amplification with expected sizes (0.8-1.7 kb). Sequencing confirmed that the products covered the entire genome of the RVFV strains tested. Primer specificity was confirmed via in silico comparison against all non-redundant nucleotide sequences using the BLASTn alignment tool in the NCBI database. To assess the clinical applicability of the primers, mock clinical specimens containing human and RVFV RNAs were prepared. The entire RVFV genome was successfully amplified and sequenced at a viral concentration of 108 copies/mL. Given the universality, specificity, and clinical applicability of the primers, we anticipate that the RVFV universal primer pairs and the developed method will aid in RVFV phylogenomics and mutation detection.

Subspecies-level genome comparison of Lactobacillus delbrueckii.

Lactobacillus delbrueckii comprises six subspecies, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. jakobsenii, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. sunkii, and L. delbrueckii subsp. indicus. We investigated the evolution of the six subspecies of L. delbrueckii using comparative genomics. While the defining feature of the species was the gene number increment driven by mobile elements and gene fragmentation, the repertoire of subspecies-specific gene gains and losses differed among the six subspecies. The horizontal gene transfer analyses indicated that frequent gene transfers between different subspecies had occurred when the six subspecies first diverged from the common ancestor, but recent gene exchange was confined to a subspecies implying independent evolution of the six subspecies. The subspecies bulgaricus is a homogeneous group that diverged from the other subspecies a long time ago and underwent convergent evolution. The subspecies lactis, jakobsenii, delbrueckii, and sunkii were more closely related to each other than to other subspecies. The four subspecies commonly show increasing genetic variability with increasing genome size. However, the four subspecies were distinguished by specific gene contents. The subspecies indicus forms a branch distant from the other subspecies and shows an independent evolutionary trend. These results could explain the differences in the habitat and nutritional requirements of the subspecies of L. delbrueckii.

Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2.

As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.

Peripheral blood transcriptomic clusters uncovered immune phenotypes of asthma.

BACKGROUND: Transcriptomic analysis has been used to elucidate the complex pathogenesis of heterogeneous disease and may also contribute to identify potential therapeutic targets by delineating the hub genes. This study aimed to investigate whether blood transcriptomic clustering can distinguish clinical and immune phenotypes of asthmatics, and microbiome in asthmatics. METHODS: Transcriptomic expression of peripheral blood mononuclear cells (PBMCs) from 47 asthmatics and 21 non-asthmatics was measured using RNA sequencing. A hierarchical clustering algorithm was used to classify asthmatics. Differentially expressed genes, clinical phenotypes, immune phenotypes, and microbiome of each transcriptomic cluster were assessed. RESULTS: In asthmatics, three distinct transcriptomic clusters with numerously different transcriptomic expressions were identified. The proportion of severe asthmatics was highest in cluster 3 as 73.3%, followed by cluster 2 (45.5%) and cluster 1 (28.6%). While cluster 1 represented clinically non-severe T2 asthma, cluster 3 tended to include severe non-T2 asthma. Cluster 2 had features of both T2 and non-T2 asthmatics characterized by the highest serum IgE level and neutrophil-dominant sputum cell population. Compared to non-asthmatics, cluster 1 showed higher CCL23 and IL1RL1 expression while the expression of TREML4 was suppressed in cluster 3. CTSD and ALDH2 showed a significant positive linear relationship across three clusters in the order of cluster 1 to 3. No significant differences in the diversities of lung and gut microbiomes were observed among transcriptomic clusters of asthmatics and non-asthmatics. However, our study has limitations in that small sample size data were analyzed with unmeasured confounding factors and causal relationships or function pathways were not verified. CONCLUSIONS: Genetic clustering based on the blood transcriptome may provide novel immunological insight, which can be biomarkers of asthma immune phenotypes. Trial registration Retrospectively registered.

Effect of luxS encoding a synthase of quorum-sensing signal molecule AI-2 of Vibrio vulnificus on mouse gut microbiome.

Autoinducer-2 (AI-2), a quorum-sensing signal molecule from the human pathogen Vibrio vulnificus, was assessed for its effect on the gut microbiome of mice. For this, we employed 16S rRNA sequencing to compare the gut microbiome of mice infected with either wild-type V. vulnificus or with the isotype ΔluxS that has a deletion in luxS which encodes the biosynthetic function of AI-2. The relative ratio of wild-type Vibrio species in the jejunum and ileum of mice infected with the wild type was significantly higher than that in mice infected with ΔluxS, suggesting that AI-2 plays an important role in the colonization of V. vulnificus in the small intestine. The bacterial composition in the gut of mice infected with ΔluxS comprises a higher proportion of Firmicutes, composed mainly of Lactobacillus, compared to the mice infected with wild-type cells. In the large intestine, Vibrio species were barely detected regardless of genetic background. Three Lactobacillus spp. isolated from fecal samples from mice infected with ΔluxS manifested significant antibacterial activities against V. vulnificus. Culture supernatants from these three species were dissolved by HPLC, and a substance in fractions showing inhibitory activity against V. vulnificus was determined to be lactic acid. Our results suggest that luxS in V. vulnificus affects not only the ability of the species to colonize the host gut but also its susceptibility to the growth-inhibiting activity of commensal bacteria including Lactobacillus. KEY POINTS: • Gut microbiomes of ΔluxS-infected and WT Vibrio-infected mice differed greatly. • Difference was most prominent in the jejunum and ileum compared to the duodenum or large intestine. • In the small and large intestines of mice, the relative proportions of Vibrio and Lactobacillus species showed a negative relationship. • Effector molecules produced by Lactobacillus in mouse gut inhibit Vibrio growth.

Human Alphacoronavirus Universal Primers for Genome Amplification and Sequencing.

Rapid and accurate sequencing covering the entire genome is essential to identify genetic variations of viral pathogens. However, due to the low viral titers in clinical samples, certain amplification steps are required for viral genome sequencing. At present, there are no universal primers available for alphacoronaviruses and that, since these viruses have diverse strains, new primers specific to the target strain must be continuously developed for sequencing. Thus, in this study, we aimed to develop a universal primer set valid for all human alphacoronaviruses and applicable to samples containing trace amounts of the virus. To this aim, we designed overlapping primer pairs capable of amplifying the entire genome of all known human alphacoronaviruses. The selected primers, named the AC primer set, were composed of 10 primer pairs stretching over the entire genome of alphacoronaviruses, and produced PCR products of the expected size (3-5 kb) from both the HCoV-229E and HCoV-NL63 strains. After genome amplification, an evaluation using various sequencing platforms was carried out. The amplicon library sequencing data were assembled into complete genome sequences in all sequencing strategies examined in this study. The sequencing accuracy varied depending on the sequencing technology, but all sequencing methods showed a sequencing error of less than 0.01%. In the mock clinical specimen, the detection limit was 10-3 PFU/ml (102 copies/ml). The AC primer set and experimental procedure optimized in this study may enable the fast diagnosis of mutant alphacoronaviruses in future epidemics.

Association of obstructive sleep apnea severity with the composition of the upper airway microbiome.

STUDY OBJECTIVES: Although the airway mucosal system plays a pivotal role in the pathogenesis of obstructive sleep apnea (OSA), the underlying disease mechanism remains unclear. The microbiome greatly impacts human health and disease, particularly in the mucosa, where it can have direct interactions. In this study, we aimed to analyze the microbiome composition in the upper airway mucosa of individuals with and without OSA to identify potential disease severity-related microbial signatures. METHODS: This population-based cohort study involved 92 participants (mean age = 62.7 ± 5.8 years; male-to-female ratio = 0.74) who underwent a physical examination and sleep study. Upper airway swab samples were collected from the nasopharyngeal mucosa to evaluate the microbiome based on 16S rRNA gene pyrosequencing. The relationship between microbiome composition and sleep parameters was explored through bioinformatics analysis. RESULTS: The average apnea-hypopnea index was 7.75 ± 6.5 events/h. Proteobacteria, Firmicutes, and Actinobacteria were the predominant phyla in the nasopharyngeal microbiota in all participants. Simpson diversity indexes were higher in patients with OSA (0.6435 ± 0.2827) than in the control patients (0.6095 ± 0.2683); however, the difference was not significant (P = .1155). Specific anaerobes negatively correlated with the lowest oxygen saturation level during sleep (sum of powered score (1) = -117.47; P = .0052). CONCLUSIONS: The upper airway microbiome of older patients with mild-moderate OSA exhibited minor differences in composition compared with that of individuals without OSA, possibly owing to environmental changes in the upper airway mucosa resulting from recurrent airway obstruction and intermittent hypoxia in patients with OSA. CITATION: Hong S-N, Kim KJ, Baek M-G, et al. Association of obstructive sleep apnea severity with the composition of the upper airway microbiome. J Clin Sleep Med. 2022;18(2):505-515.

Distribution of bacteriocin genes in the lineages of Lactiplantibacillus plantarum.

Lactiplantibacillus plantarum, previously named "Lactobacillus plantarum," is found in a wide variety of environments exhibiting a high level of intraspecies genetic diversity. To investigate the strain diversity, we performed comparative genomic analyses of the 54 complete genome sequences. The results revealed that L. plantarum subsp. plantarum was split into three lineages, A, B and C. Of the genes beneficial for probiotic activity, only those associated with the biosynthesis of plantaricin (Pln), an L. plantarum-specific bacteriocin, were found to be significantly different among the lineages. The genes related to the biosynthesis of plnE/F were conserved throughout the three lineages, whereas the outgroups did not possess any Pln-producing genes. In lineage C, the deepest and ancestral type branch, plnE/F genes, were well conserved. In lineage B, loss of gene function was observed due to mobile elements in the pln loci. In lineage A, most strains were predicted to produce more than one type of Pln by possessing diverse Pln-encoding genes. These results showed the presence of functional diversity arising from the trifurcating evolution in L. plantarum subsp. plantarum and demonstrated that Pln is an indicator for differentiating the three lineages.

Modulation of the Gut Microbiota by Sihocheonggan-Tang Shapes the Immune Responses of Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by complex immune dysregulation and closely related to the gut microbiome. The present study investigated the microbiome-mediated effect of Sihocheonggan-Tang (SHCGT) on AD-like symptoms induced by 2,4-dinitrochlorobenzene (DNCB) in BALB/c mice. DNCB was applied regularly to the ear and dorsal skin of BALB/c mice, and SHCGT was administered orally daily for 2 weeks. The composition of the gut microbiota was analyzed using 16S rRNA sequencing, and the effect of gut microbiome-derived metabolites, specifically short-chain fatty acids (SCFAs), was evaluated in tumor necrosis factor-alpha (TNF-α)- and interferon-gamma (IFN-γ)-treated HaCaT cells. SHCGT alleviated DNCB-induced symptoms of AD and the immune response to AD by decreasing the plasma immunoglobulin E level and splenic interleukin-4, interleukin-10, TNF-α, and IFN-γ levels. The gut microbiome composition and the damaged gut epithelial barrier in mice with AD were also significantly altered by SHCGT, and the reduced SCFA levels therein were elevated. We found that SFCAs directly inhibited the mRNA expression of IL-6 and ICAM-1 in TNF-α- and INF-γ-treated HaCaT cells. The finding that SHCGT regulates the gut microbiome and improves DNCB-induced AD in mice suggests that this herbal medicine has therapeutic potential in patients with AD.

VicPred: A Vibrio cholerae Genotype Prediction Tool.

Genomic information can be used to predict major pathogenic traits of pathogens without the need for laboratory experimentation. However, no Vibrio cholerae genome-based trait identification tools currently exist. The aim of this study was to develop a web-based prediction tool to identify Vibrio pathogenic traits using publicly available 796 whole-genome sequences of V. cholerae. Using this application, 68 structural O-antigen gene clusters belonging to 49 serogroups of V. cholerae were classified, and the composition of the genes within the O-antigen cluster of each serogroup was identified. The arrangement and location of the CTX prophage and related elements of the seventh cholera pandemic strains were also revealed. With the versatile tool, named VicPred, we analyzed the assemblage of various SXTs (sulfamethoxazole/trimethoprim resistance element) and major genomic islands (GIs) of V. cholerae, and the increasing trend in drug-resistance revealing high resistance of the V. cholerae strains to certain antibiotics. The pathogenic traits of newly sequenced V. cholerae strains could be analyzed based on these characteristics. The accumulation of further genome data will expedite the establishment of a more precise genome-based pathogenic traits analysis tool.

Interactions between NCR+ILC3s and the Microbiome in the Airways Shape Asthma Severity.

Asthma is a heterogeneous disease whose development is shaped by a variety of environmental and genetic factors. While several recent studies suggest that microbial dysbiosis in the gut may promote asthma, little is known about the relationship between the recently discovered lung microbiome and asthma. Innate lymphoid cells (ILCs) have also been shown recently to participate in asthma. To investigate the relationship between the lung microbiome, ILCs, and asthma, we recruited 23 healthy controls (HC), 42 patients with non-severe asthma, and 32 patients with severe asthma. Flow cytometry analysis showed severe asthma associated with fewer natural cytotoxicity receptor (NCR)+ILC3s in the lung. Similar changes in other ILC subsets, macrophages, and monocytes were not observed. The asthma patients did not differ from the HC in terms of the alpha and beta-diversity of the lung and gut microbiomes. However, lung function correlated positively with both NCR+ILC3 frequencies and microbial diversity in the lung. Sputum NCR+ILC3 frequencies correlated positively with lung microbiome diversity in the HC, but this relationship was inversed in severe asthma. Together, these data suggest that airway NCR+ILC3s may contribute to a healthy commensal diversity and normal lung function.

Microbiome profiling of uncinate tissue and nasal polyps in patients with chronic rhinosinusitis using swab and tissue biopsy.

Chronic rhinosinusitis (CRS) is characterized according to the presence or absence of nasal polyps (NPs) and displays nasal microbiota dysbiosis. However, optimal sampling methods of the nasal microbiome in CRS have not been identified. We aimed to assess the microbial composition in patients with CRS, comparing different sampling methods (swab and tissue biopsy), tissue types (uncinate tissue and NP), and disease subtypes. Samples were obtained by swabbing the middle meatus and taking a biopsy of uncinate tissue (UT) in patients with CRS with (CRSwNP, N = 8) or without NP (CRSsNP, N = 6) and controls (N = 8). NPs were also harvested in CRSwNP. DNAs were extracted from fifty-two samples and analyzed by 16S rRNA gene amplicon sequencing. As a result, a great interpersonal variance was observed in nasal swabs, while UT samples presented distinct microbiome with low inter-personal differences. Moreover, the UT microbiomes were further differentiated into three clusters which are associated with disease status (control, CRSsNP, and CRSwNP). Compared to UT, NP revealed a unique microbiome profile with significantly less bacterial diversity. Prevotella was the genus whose abundance was negatively correlated with disease severity in NP. In conclusion, tissue samples are better specimens than nasal swabs for assessing the microbiomes of CRS patients. Several bacteria in UT and NP tissues revealed an association with clinical severity of CRSwNP.

Lung virome: New potential biomarkers for asthma severity and exacerbation.

BACKGROUND: Although some respiratory virus infections are known to contribute to the development and exacerbation of asthma, commensal viromes in airway have not been extensively studied due to technical challenges. OBJECTIVES: This study investigated the characteristics of the virome in asthmatic airways. METHODS: Both the bacteriome and virome profiles in sputum from 12 healthy individuals, 15 patients with nonsevere asthma, and 15 patients with severe asthma were analyzed and assessed for the association with clinical characteristics such as severity, exacerbation, Asthma Control Test (ACT), and lung function. RESULTS: While analysis of the 16S ribosomal RNA bacteriome in the airway showed no differences, clear contrasts in the diversity and composition of airway viromes were observed between healthy controls and patients with asthma. Herpesviruses were the most abundant type of virus in the asthma group (44.6 ± 4.6%), mainly with cytomegalovirus (CMV) and EBV accounting for 24.5 ± 3.3% and 16.9 ± 3.5%, respectively, in contrast to those in the healthy controls (5.4 ± 2.5% and 7.1 ± 3.0%, respectively). CMV and EBV were more abundant in patients with asthma who experienced exacerbation, and their abundance showed correlation with more severe asthma, lower ACT score, and lower lung function. On the contrary, bacteriophage that is abundant in healthy controls was severely reduced in patients with asthma in the order of nonsevere and severe asthma and presented significant positive correlation with ACT and FEV1/forced vital capacity. CONCLUSIONS: Lung viromes, especially, CMV, EBV, and bacteriophage may be potential biomarkers of asthma severity and exacerbation.

A Half-Day Genome Sequencing Protocol for Middle East Respiratory Syndrome Coronavirus.

Recent coronavirus (CoV) outbreaks, including that of Middle East respiratory syndrome (MERS), have presented a threat to public health worldwide. A primary concern in these outbreaks is the extent of mutations in the CoV, and the content of viral variation that can be determined only by whole genome sequencing (WGS). We aimed to develop a time efficient WGS protocol, using universal primers spanning the entire MERS-CoV genome. MERS and synthetic Neoromicia capensis bat CoV genomes were successfully amplified using our developed PCR primer set and sequenced with MinION. All experimental and analytical processes took 6 h to complete and were also applied to synthetic animal serum samples, wherein the MERS-CoV genome sequence was completely recovered. Results showed that the complete genome of MERS-CoV and related variants could be directly obtained from clinical samples within half a day. Consequently, this method will contribute to rapid MERS diagnosis, particularly in future CoV epidemics.

Poseidonibacter parvus sp. nov., isolated from a squid.

A Gram-stain-negative, aerobic, short rod-shaped, motile, brownish-coloured bacterium, termed strain LPB0137T, was isolated from a squid. Its cells could grow weakly on marine agar 2216 with 0.04 % 2,3,5-triphenyl tetrazolium chloride (TTC). Each cell of strain LPB0137T has a circular chromosome with a length of 2.87 Mb and 27.7 mol% DNA G+C content. The genome includes 2698 protein-coding genes and six rRNA operons. In 16S rRNA gene sequence trees, strain LPB0137T formed a robust monophyletic clade with Poseidonibacter antarcticus SM1702T with a sequence similarity of 98.3 %. However, the average nucleotide identity and in silico DNA-DNA hybridization values between the two type strains were low (83.9 and 28.1 %, respectively). The overall phenotypic and genomic features of strain LPB0137T supported its assignment to the genus Poseidonibacter. However, the relatively low gene and genome sequence similarity between this strain and other type strains of the genus Poseidonibacter and several enzymatic characteristics indicated the taxonomic novelty of the isolated strain as a new member of the genus Poseidonibacter. Therefore, based on the phylogenetic and phenotypic characteristics of LPB0137T, we proposed a novel species of the genus Poseidonibacter for it, with the name Poseidonibacter parvus sp. nov. The type strain of this new species is thus LPB0137T (=KACC 18888T=JCM 31548T).

Bacterial Signatures of Paediatric Respiratory Disease: An Individual Participant Data Meta-Analysis.

Introduction: The airway microbiota has been linked to specific paediatric respiratory diseases, but studies are often small. It remains unclear whether particular bacteria are associated with a given disease, or if a more general, non-specific microbiota association with disease exists, as suggested for the gut. We investigated overarching patterns of bacterial association with acute and chronic paediatric respiratory disease in an individual participant data (IPD) meta-analysis of 16S rRNA gene sequences from published respiratory microbiota studies. Methods: We obtained raw microbiota data from public repositories or via communication with corresponding authors. Cross-sectional analyses of the paediatric (<18 years) microbiota in acute and chronic respiratory conditions, with >10 case subjects were included. Sequence data were processed using a uniform bioinformatics pipeline, removing a potentially substantial source of variation. Microbiota differences across diagnoses were assessed using alpha- and beta-diversity approaches, machine learning, and biomarker analyses. Results: We ultimately included 20 studies containing individual data from 2624 children. Disease was associated with lower bacterial diversity in nasal and lower airway samples and higher relative abundances of specific nasal taxa including Streptococcus and Haemophilus. Machine learning success in assigning samples to diagnostic groupings varied with anatomical site, with positive predictive value and sensitivity ranging from 43 to 100 and 8 to 99%, respectively. Conclusion: IPD meta-analysis of the respiratory microbiota across multiple diseases allowed identification of a non-specific disease association which cannot be recognised by studying a single disease. Whilst imperfect, machine learning offers promise as a potential additional tool to aid clinical diagnosis.

Respiratory microbiome profiles differ by recent hospitalization and nursing home residence in patients on mechanical ventilation.

BACKGROUND: Healthcare-associated pneumonia (HCAP) is a heterogeneous disease. We redefined nursing-home- and hospital-associated infections (NHAI) group by revising existing HCAP risk factors. The NHAI group comprised nursing home residents with a poor functional status, or recent (past 90 days) hospitalization or recent (past 180 days) antibiotic therapy. Our aim was to determine whether respiratory microbiota profiles are related to newly defined NHAI group in critically ill patients on mechanical ventilation. METHODS: The 180 endotracheal aspirates (ETAs) from 60 mechanically ventilated ICU patients (NHAI group, n = 24; non-NHAI group, n = 36) were prospectively collected on days 1, 3 and 7 in a university hospital. The bacterial community profiles of the ETAs were explored by 16S rRNA gene sequencing. A phylogenetic-tree-based microbiome association test (TMAT), generalized linear mixed models (GLMMs), the Wilcoxon test and the reference frame method were used to analyze the association between microbiome abundance and disease phenotype. RESULTS: The relative abundance of the genus Corynebacterium was significantly higher in the pneumonia than in the non-pneumonia group. The microbiome analysis revealed significantly lower α-diversity in the NHAI group than in the non-NHAI group. In the analysis of ß-diversity, the structure of the microbiome also differed significantly between the two groups (weighted UniFrac distance, Adonis, p < 0.001). The abundance of Corynebacterium was significantly higher, and the relative abundances of Granulicatella, Staphylococcus, Streptococcus and Veillonella were significantly lower, in the NHAI group than in the non-NHAI group. CONCLUSIONS: The microbiota signature of the ETAs distinguished between patients with and without risk factors for NHAI. The lung microbiome may serve as a therapeutic target for NHAI group.