BACKGROUND: Our understanding of airway dysbiosis in chronic obstructive pulmonary disease (COPD) remains incomplete, which may be improved by unraveling the complexity in microbial interactome.
OBJECTIVE: To characterize reproducible features of airway bacterial interactome in COPD at clinical stability and during exacerbation, and evaluate their associations with disease phenotypes.
METHODS: We performed weighted ensemble-based co-occurrence network analysis of 1742 sputum microbiomes from published and new microbiome datasets, comprising two case-control studies of stable COPD versus healthy control, two studies of COPD stability versus exacerbation, and one study with exacerbation-recovery time series data.
RESULTS: Patients with COPD had reproducibly lower degree of negative bacterial interactions, i.e. total number of negative interactions as a proportion of total interactions, in their airway microbiome compared with healthy controls. Evaluation of the Haemophilus interactome showed that the antagonistic interaction networks of this established pathogen rather than its abundance consistently changed in COPD. Interactome dynamic analysis revealed reproducibly reduced antagonistic interactions but not diversity loss during COPD exacerbation, which recovered after treatment. In phenotypic analysis, unsupervised network clustering showed that loss of antagonistic interactions was associated with worse clinical symptoms (dyspnea), poorer lung function, exaggerated neutrophilic inflammation, and higher exacerbation risk. Furthermore, the frequent exacerbators (≥ 2 exacerbations per year) had significantly reduced antagonistic bacterial interactions while exhibiting subtle compositional changes in their airway microbiota.
CONCLUSIONS: Bacterial interactome disturbance characterized by reduced antagonistic interactions, rather than change in pathogen abundance or diversity, is a reproducible feature of airway dysbiosis in COPD clinical stability and exacerbations, which suggests that we may target interactome rather than pathogen alone for disease treatment.

The prevalence and clinical correlates of antibiotic resistance genes (ARGs) in bronchiectasis are not entirely clear. We aimed to profile the ARGs in sputum from adults with bronchiectasis, and explore the association with airway microbiome and disease severity and subtypes. In this longitudinal study, we prospectively collected 118 sputum samples from stable and exacerbation visits of 82 bronchiectasis patients and 19 healthy subjects. We profiled ARGs with shotgun metagenomic sequencing, and linked these to sputum microbiome and clinical characteristics, followed by validation in an international cohort. We compared ARG profiles in bronchiectasis according to disease severity, blood and sputum inflammatory subtypes. Unsupervised clustering revealed a Pseudomonas predominant subgroup (n = 16), Haemophilus predominant subgroup (n = 48), and balanced microbiome subgroup (N = 54). ARGs of multi-drug resistance were over-dominant in the Pseudomonas-predominant subgroup, while ARGs of beta-lactam resistance were most abundant in the Haemophilus-predominant subgroup. Pseudomonas-predominant subgroup yielded the highest ARG diversity and total abundance, while Haemophilus-predominant subgroup and balanced microbiota subgroup were lowest in ARG diversity and total abundance. PBP-1A, ksgA and emrB (multidrug) were most significantly enriched in Haemophilus-predominant subtype. ARGs generally correlated positively with Bronchiectasis Severity Index, fluoroquinolone use, and modified Reiff score. 68.6% of the ARG-clinical correlations could be validated in an independent international cohort. In conclusion, ARGs are differentially associated with the dominant microbiome and clinical characteristics in bronchiectasis.

Haemophilus seminalis is a newly proposed species that is phylogenetically related to Haemophilus haemolyticus. The distribution of H. seminalis in the human population, its genomic diversity, and its pathogenic potential are still unclear. This study reports the finding of our comparative genomic analyses of four newly isolated Haemophilus strains (SZY H8, SZY H35, SZY H36, and SZY H68) from human sputum specimens (Guangzhou, China) along with the publicly available genomes of other phylogenetically related Haemophilus species. Based on pairwise comparisons of the 16S rRNA gene sequences, the four isolates showed <98.65% sequence identity to the type strains of all known Haemophilus species but were identified as belonging to H. seminalis, based on comparable phenotypic and genotypic features. Additionally, the four isolates showed high genome-genome relatedness indices (>95% ANI values) with 17 strains that were previously identified as either \"Haemophilus intermedius\" or hemin (X-factor)-independent H. haemolyticus and therefore required a more detailed classification study. Phylogenetically, these isolates, along with the two previously described H. seminalis isolates (a total of 23 isolates), shared a highly homologous lineage that is distinct from the clades of the main H. haemolyticus and Haemophilus influenzae strains. These isolates present an open pangenome with multiple virulence genes. Notably, all 23 isolates have a functional heme biosynthesis pathway that is similar to that of Haemophilus parainfluenzae. The phenotype of hemin (X-factor) independence and the analysis of the ispD, pepG, and moeA genes can be used to distinguish these isolates from H. haemolyticus and H. influenzae. Based on the above findings, we propose a reclassification for all \"H. intermedius\" and two H. haemolyticus isolates belonging to H. seminalis with an emended description of H. seminalis. This study provides a more accurate identification of Haemophilus isolates for use in the clinical laboratory and a better understanding of the clinical significance and genetic diversity in human environments. IMPORTANCE As a versatile opportunistic pathogen, the accurate identification of Haemophilus species is a challenge in clinical practice. In this study, we characterized the phenotypic and genotypic features of four H. seminalis strains that were isolated from human sputum specimens and propose the \"H. intermedius\" and hemin (X-factor)-independent H. haemolyticus isolates as belonging to H. seminalis. The prediction of virulence-related genes indicates that H. seminalis isolates carry several virulence genes that are likely to play an important role in its pathogenicity. In addition, we depict that the genes ispD, pepG, and moeA can be used as biomarkers for distinguishing H. seminalis from H. haemolyticus and H. influenzae. Our findings provide some insights into the identification, epidemiology, genetic diversity, pathogenic potential, and antimicrobial resistance of the newly proposed H. seminalis.

Increasing evidence indicates that respiratory tract microecological disorders may play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Understanding the composition of the respiratory microbiome in COPD and its relevance to respiratory immunity will help develop microbiome-based diagnostic and therapeutic approaches. One hundred longitudinal sputum samples from 35 subjects with acute exacerbation of COPD (AECOPD) were analysed for respiratory bacterial microbiome using 16S ribosomal RNA amplicon sequencing technology, and the sputum supernatant was analysed for 12 cytokines using a Luminex liquid suspension chip. Unsupervised hierarchical clustering was employed to evaluate the existence of distinct microbial clusters. In AECOPD, the respiratory microbial diversity decreased, and the community composition changed significantly. The abundances of Haemophilus, Moraxella, Klebsiella, and Pseudomonas increased significantly. Significant positive correlations between the abundance of Pseudomonas and TNF-α, abundance of Klebsiella and the percentage of eosinophils were observed. Furthermore, COPD can be divided into four clusters based on the respiratory microbiome. AECOPD-related cluster was characterized by the enrichment of Pseudomonas and Haemophilus and a high level of TNF-α. Lactobacillus and Veillonella are enriched in therapy-related phenotypes and may play potential probiotic roles. There are two inflammatory endotypes in the stable state: Gemella is associated with the Th2 inflammatory endotypes, whereas Prevotella is associated with the Th17 inflammatory endotypes. Nevertheless, no differences in clinical manifestations were found between these two endotypes. The sputum microbiome is associated with the disease status of COPD, allowing us to distinguish different inflammatory endotypes. Targeted anti-inflammatory and anti-infective therapies may improve the long-term prognosis of COPD.

Introduction: Nemonoxacin is an innovative quinolone antibiotic for treatment of community-acquired pneumonia (CAP). As more data are available from clinical studies, it is necessary to perform an integrative pharmacokinetic/pharmacodynamic (PK/PD) analysis to support and justify the optimal dosing regimen of nemonoxacin in clinical practice. Methods and Results: We developed a population PK model using non-linear mixed effect model based on the data of 195 Chinese subjects receiving nemonoxacin in phase I to III clinical trials. The base model was a standard two-compartment PK model defined by clearance (12 L/h) and central volume of distribution (86 L). Covariates included creatinine clearance (CLcr), body weight (BW), sex, disease status and food. Compared to the subject with BW 60 kg, Cmax and A U C 0 - 24 ,   ss reduced by 24% and 19% in the subject with BW 80 kg, respectively. Compared to the subject with CLcr 150 ml/min, A U C 0 - 24 ,   ss and T1/2 increased by 28% and 24%, respectively in the subject with CLcr 30 ml/min. Compared to the fasted status, Tmax of nemonoxacin increased by 1.2 h in the subject with fed status. Effects of sex and disease status on PK parameters were small (change of PK parameters ≤19%). AUC0-24/MIC and %T > MIC were identified as the optimal PK/PD indices for predicting clinical efficacy. The AUC0-24/MIC target was 63.3, 97.8, and 115.7 against Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae, respectively. The %T > MIC target was 7.96% against Klebsiella pneumoniae. Monte Carlo simulation showed that treatment with nemonoxacin 500 mg q24 h could attain a PK/PD cutoff value higher than the MIC90 against S. pneumoniae and S. aureus. The corresponding cumulative fraction of response (CFR) was greater than 93%, while nemonoxacin 750 mg q24 h would provide higher PK/PD cutoff value against Haemophilus parainfluenzae, and higher CFR (83%) than 500 mg q24 h. Conclusion: Integrative PK/PD analysis justifies the reliable clinical and microbiological efficacy of nemonoxacin 500 mg q24 h in treating CAP caused by S. pneumoniae, S. aureus, and K. pneumoniae, irrespective of patient sex, mild renal impairment, empty stomach or not. However, nemonoxacin 750 mg q24 h would provide better efficacy than 500 mg q24 h for the CAP caused by H. parainfluenzae in terms of CFR.

To investigate the lipid profiles and intestinal microflora in pregnant patients with hypothyroidism and their correlation with pregnancy outcomes.
In total, 27 pregnant women with hypothyroidism (study case) and 28 normal pregnant women (control group) were enrolled in this study. The lipid profiles and intestinal microflora in the two groups were compared using untargeted liquid chromatography-mass spectrometry (LC-MS) and 16S rRNA amplicon sequencing, respectively. The association among the differential metabolites, intestinal microflora, serological indicators and pregnancy outcomes was further analyzed.
Patients in study case had higher C-reactive protein (CRP) levels (P = 0.025) and lower birth weight (P=0.005) than the control group. A total of 42 differential lipid metabolites and 7 enrichment KEGG pathways were obtained between the two groups (VIP ≥ 1, P < 0.05). Ten lipid metabolites can be used as characteristic metabolites of study case, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM). The richness and diversity of intestinal microflora in study case were lower than those in the control group (P>0.05). LEfSe analysis revealed that patients in study case had higher abundance of Prevotella and Haemophilus and lower abundance of Blautia than the control group (P < 0.05). Blautia was positively correlated with SM and negatively correlated with PC and PE; the CRP level and Prevotella were positively correlated; the neonatal weight and PC level were negatively correlated (P < 0.05).
The lipid profile and intestinal microflora of pregnant women with hypothyroidism significantly differed from those of normal pregnant women and were associated with adverse pregnancy outcomes. The interaction between lipid metabolism and intestinal microflora may be a potential target for further studies investigating the pathogenesis of hypothyroidism during pregnancy.

Many factors affecting satellitism tests are unclear, and it is difficult to avoid misidentification, even if the medium is properly selected. We investigated the factors causing false-positive results for Haemophilus influenzae and false-negative results for Haemophilus parainfluenzae in the satellitism tests using Staphylococcus aureus as the source of nicotinamide adenine dinucleotide (NAD). H. influenzae (four reference strains and 47 clinical isolates), H. parainfluenzae (two reference strains and 67 clinical isolates), four different media, and two strains of S. aureus revived on two different media were used in this study. The type of medium used to revive S. aureus was the most common factor causing false-positive results for H. influenzae, followed by different strains of S. aureus and the type of medium used for the experiment. The production of false-negative results for H. parainfluenzae was only related to the medium used in the experiment. To improve the accuracy of the tests in routine laboratories, using S. aureus as the source of NAD, tryptic soy agar, and S. aureus (ATCC 25923) revived on nutrient agar should be adopted.

Periodontitis is a globally prevalent disease that imposes a functional and aesthetic burden on patients. The oral microbiome influences human health. The aim of this study was at assessing gender variation in the subgingival bacterial microbiome of elderly patients with initial periodontitis and to determine the causes of this variation. Twelve males and twenty females (range 50-68 years old) with initial periodontitis provided subgingival plaque samples. 16S rRNA gene sequencing, QIIME-based data processing, and statistical analyses were carried out using several different analytical approaches to detect differences in the oral microbiome between the two groups. Males had higher Chao1 index, observed species, and phylogenetic diversity whole tree values than females. Analysis of β-diversity indicated that the samples were reasonably divided by the gender. The linear discriminant analysis effect size showed that the most representative biomarkers were the genus Haemophilus in males, whereas the dominant bacteria in females were Campylobacter. Kyoto Encyclopedia of Genes and Genomes analysis showed that predicting changes in the female oral microbiota may be related to the immune system and immune system diseases are the main factor in males. These data suggest that gender may be a differentiating factor in the microbial composition of subgingival plaques in elderly patients with initial periodontitis. These results could deepen our understanding of the role of gender in the oral microbiota present during initial periodontitis.

OBJECTIVE: To investigate the salivary metaproteomic characteristics of the children with and without severe early childhood caries (S-ECC).
METHODS: In this study, we collected unstimulated saliva samples from 34 children (age 3-4 years) with caries free (NC, dmfs (= index of decayed, missing due to caries, or filled tooth surfaces) = 0, n = 23) and with S-ECC (dmfs≥10, n = 11). Salivary proteins were extracted and reduced, and then a Liquid Chromatography/Mass Spectrometry system was used to identify proteins.
RESULTS: Nearly 3000 proteins were identified in this study, and about 3.5 % of the proteins originated from human while 86 % were derived from microbes. The salivary protein types in the NC group were statistically greater than those in the S-ECC group (P <0.05). Specifically, the salivary protein types derived from microbes in the NC group were significantly greater than those in the S-ECC group. Three proteins, human lactoferrin, penicillin-binding protein 1C [Burkholderia ubonensis], human alpha-defensin 1 (F28a mutant), were decreased statistically in the NC group compared to the S-ECC group (P < 0.05). Only one protein, 50S ribosomal protein L17 secreted by Haemophilus haemolyticus, was significantly increased in the NC group compared to the S-ECC group. Salivary IgA was the top highest protein in the NC group whereas human lysozyme was the top highest protein in the S-ECC group.
CONCLUSIONS: The differential proteins recognized in this study may be conducive for finding a caries biomarker. Understanding the metaproteomic characteristics can help us to control the caries from human origin and microbial origin.

Disturbance in microbiota affects the mucosal immune response, and it is gradually recognized to be associated with the Immunoglobin A nephropathy (IgAN). This study aims to explore the potential roles of oral microbiota in disease pathogenesis. Saliva samples were collected from 31 patients with IgAN and 30 controls for 16S rRNA gene sequencing. The evenness, diversity, and composition of oral microbiota were analyzed. Moreover, sub-phenotype association analysis was conducted. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to investigate microbiota functions. Compared to healthy controls, microbial diversity tended to decrease in IgAN, and the microbial profiles were remarkably distinguished. The relative abundance of Capnocytophaga and SR1_genera_incertae_sedis were enriched, whereas 17 genera, such as Rothia, were significantly reduced in IgAN. Variable importance in projection scores showed that 12 genera, including Capnocytophaga, Rothia, and Haemophilus, could discriminate between the two groups. In the sub-phenotype correlation analysis, the relative abundance of Capnocytophaga and Haemophilus was positively associated with levels of proteinuria and serum IgA, respectively. Further metabolic pathway analysis showed 7 predictive functional profiles, including glycosphingolipid biosynthesis, oxidative phosphorylation, and N-glycan biosynthesis were enriched in IgAN. In conclusion, disturbance in oral microbiota was observed to be associated with IgAN and its sub-phenotypes, which may shed novel insights into disease pathogenesis from a microbiome perspective.