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1.
Short bowel syndrome (SBS) is observed in Humans after a large resection of gut. Since the remnant colon and its associated microbiota play a major role in the outcome of patients with SBS, we studied the overall qualitative and quantitative microbiota composition of SBS adult patients compared to controls. The population was composed of 11 SBS type II patients (with a jejuno-colonic anastomosis) and 8 controls without intestinal pathology. SBS patients had 38 ± 30 cm remnant small bowel length and 66 ± 19% of residual colon. The repartition of proteins, lipids, carbohydrates and fibres was expressed as % of total oral intake in patients and controls. The microbiota was profiled from stool and biopsy samples with temporal temperature gradient gel electrophoresis and quantitative PCR. We show here that microbiota of SBS patients is unbalanced with a high prevalence of Lactobacillus along with a sub-dominant presence and poor diversity of Clostridium leptum, Clostridium coccoides and Bacteroidetes. In addition, Lactobacillus mucosae was detected within the fecal and mucosa-associated microbiota of SBS patients, whereas it remained undetectable in controls. Thus, in SBS the microbial composition was deeply altered in fecal and mucosal samples, with a shift between dominant and sub-dominant microbial groups and the prevalence of L. mucosae.  相似文献   

2.
Analysis of microbiota in various biological and environmental samples under a variety of conditions has recently become more practical due to remarkable advances in next-generation sequencing. Changes leading to specific biological states including some of the more complex diseases can now be characterized with relative ease. It is known that gut microbiota is involved in the pathogenesis of inflammatory bowel disease (IBD), mainly Crohn''s disease and ulcerative colitis, exhibiting symptoms in the gastrointestinal tract. Recent studies also showed increased frequency of oral manifestations among IBD patients, indicating aberrations in the oral microbiota. Based on these observations, we analyzed the composition of salivary microbiota of 35 IBD patients by 454 pyrosequencing of the bacterial 16S rRNA gene and compared it with that of 24 healthy controls (HCs). The results showed that Bacteroidetes was significantly increased with a concurrent decrease in Proteobacteria in the salivary microbiota of IBD patients. The dominant genera, Streptococcus, Prevotella, Neisseria, Haemophilus, Veillonella, and Gemella, were found to largely contribute to dysbiosis (dysbacteriosis) observed in the salivary microbiota of IBD patients. Analysis of immunological biomarkers in the saliva of IBD patients showed elevated levels of many inflammatory cytokines and immunoglobulin A, and a lower lysozyme level. A strong correlation was shown between lysozyme and IL-1β levels and the relative abundance of Streptococcus, Prevotella, Haemophilus and Veillonella. Our data demonstrate that dysbiosis of salivary microbiota is associated with inflammatory responses in IBD patients, suggesting that it is possibly linked to dysbiosis of their gut microbiota.  相似文献   

3.
Ulcerative colitis (UC) is a recurrent pathology of complex etiology that has been occasionally associated with oral lesions, but the overall composition of the oral microbiome in UC patients and its role in the pathogenesis of the disease are still poorly understood. In this study, the oral microbiome of UC patients and healthy individuals was compared to ascertain the possible changes in the oral microbial communities associated with UC. For this, the salivary microbiota of 10 patients diagnosed with an active phase of UC and 11 healthy controls was analyzed by 16S rRNA gene sequencing (trial ref. ISRCTN39987). Metataxonomic analysis revealed a decrease in the alpha diversity and an imbalance in the relative proportions of some key members of the oral core microbiome in UC patients. Additionally, Staphylococcus members and four differential species or phylotypes were only present in UC patients, not being detected in healthy subjects. This study provides a global snapshot of the existence of oral dysbiosis associated with UC, and the possible presence of potential oral biomarkers.  相似文献   

4.
The investigation provides molecular analyses of the faecal microbiota in type 2 diabetic patients. In order to characterise the gut microbiota in diabetic patients and to assess whether there are changes in the diversity and similarity of gut microbiota in diabetic patients when compared with healthy individuals, bacterial DNAs from 16 type 2 diabetic patients and 12 healthy individuals were extracted from faecal samples and characterised by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specifically targeting V3 region of the 16S rRNA gene, as well as been sequenced for excised gel bands. The counts of Bacteroides vulgatus, Clostridium leptum subgroup and Bifidobacterium genus were assessed using quantitative PCR. By comparing species diversity profiles of two groups, we observed that there were no significant differences between diabetic and healthy group, although a few diabetic individuals (D6, D8) exhibited a remarkable decrease in species profiles. As for the similarity index, it was lower in inter-group than that in intra-group, which showed that the composition of gut microbiota in diabetic group might be changed due to diabetes status. Sequencing results also revealed that bacterial composition of diabetic group was different from that of the healthy group. B. vulgatus and Bifidobacterium genus were low represented in the microbiota of diabetic group, and the significant decrease was observed for Bifidobacterium by real-time PCR. Taken together, in this work we observed the characterisation of gut microbiota in diabetic patients, which suggestes that the gut microbiota of diabetes patients have some changes associated with occurrence and development of diabetes.  相似文献   

5.
The coronary artery disease (CAD) is a chronic inflammatory disease involving genetic as well as environmental factors. Recent evidence suggests that the oral microbiome has a significant role in triggering atherosclerosis. The present study assessed the oral microbiome composition variation between coronary patients and healthy subjects in order to identify a potential pathogenic signature associated with CAD. We performed metagenomic profiling of salivary microbiomes by 16S ribosomal RNA (rRNA) next-generation sequencing. Oral microbiota profiling was performed for 30 individuals including 20 patients with CAD and ten healthy individuals without carotid plaques or previous stroke or myocardial infarction.We found that oral microbial communities in patients and healthy controls are represented by similar global core oral microbiome. The predominant taxa belonged to Firmicutes (genus Streptococcus, Veillonella, Granulicatella, Selenomonas), Proteobacteria (genus Neisseria, Haemophilus), Actinobacteria (genus Rothia), Bacteroidetes (genus Prevotella, Porphyromonas), and Fusobacteria (genus Fusobacterium, Leptotrichia). More than 60% relative abundance of each sample for both CAD patients and controls is represented by three major genera including Streptococcus (24.97 and 26.33%), Veillonella (21.43 and 19.91%), and Neisseria (14.23 and 15.33%).Using penalized regression analysis, the bacterial genus Eikenella was involved as the major discriminant genus for both status and Syntax score of CAD. We also reported a significant negative correlation between Syntax score and Eikenella abundance in coronary patients’ group (Spearman rho = −0.68, P=0.00094).In conclusion, the abundance of Eikenella in oral coronary patient samples compared with controls could be a prominent pathological indicator for the development of CAD.  相似文献   

6.
Saliva is a biofluid that maintains the health of oral tissues and the homeostasis of oral microbiota. Studies have demonstrated that Oral squamous cell carcinoma (OSCC) patients have different salivary microbiota than healthy individuals. However, the relationship between these microbial differences and clinicopathological outcomes is still far from conclusive. Herein, we investigate the capability of using metagenomic and metaproteomic saliva profiles to distinguish between Control (C), OSCC without active lesion (L0), and OSCC with active lesion (L1) patients. The results show that there are significantly distinct taxonomies and functional changes in L1 patients compared to C and L0 patients, suggesting compositional modulation of the oral microbiome, as the relative abundances of Centipeda, Veillonella, and Gemella suggested by metagenomics are correlated with tumor size, clinical stage, and active lesion. Metagenomics results also demonstrated that poor overall patient survival is associated with a higher relative abundance of Stenophotromonas, Staphylococcus, Centipeda, Selenomonas, Alloscordovia, and Acitenobacter. Finally, compositional and functional differences in the saliva content by metaproteomics analysis can distinguish healthy individuals from OSCC patients. In summary, our study suggests that oral microbiota and their protein abundance have potential diagnosis and prognosis value for oral cancer patients. Further studies are necessary to understand the role of uniquely detected metaproteins in the microbiota of healthy and OSCC patients as well as the crosstalk between saliva host proteins and the oral microbiome present in OSCC.  相似文献   

7.
Describing the biogeography of bacterial communities within the human body is critical for establishing healthy baselines from which to detect differences associated with diseases. Little is known, however, about the baseline of normal salivary microbiota from healthy Chinese children and adults. With parallel barcoded 454 pyrosequencing, the bacterial diversity and richness of saliva were thoroughly investigated from ten healthy Chinese children and adults. The overall taxonomic distribution of our metagenomic data demonstrated that the diversity of salivary microbiota from children was more complex than adults, while the composition and richness of salivary microbiota were similar in children and adults, especially for predominant bacteria. A large number of bacterial phylotypes were shared by healthy children and adults, indicating the existence of a core salivary microbiome. In children and adults, the vast majority of sequences in salivary microbiota belonged to Streptococcus, Prevotella, Neisseria, Haemophilus, Porphyromonas, Gemella, Rothia, Granulicatella, Fusobacterium, Actinomyces, Veillonella, and Aggregatibacter, which constituted the major components of normal salivary microbiota. With the exception of Actinomyces, the other seven non-predominant bacteria including Moraxella, Leptotrichia, Peptostreptococcus, Eubacterium, and members of Neisseriaceae, Flavobacteriaceae, and SR1 showed significant differences between children and adults (p?<?0.05). We first established the framework of normal salivary microbiota from healthy Chinese children and adults. Our data represent a critical step for determining the diversity of healthy microbiota in Chinese children and adults, and our data established a platform for additional large-scale studies focusing on the interactions between health and diseases in the future.  相似文献   

8.
9.
Poor oral health has been linked with an increased risk of esophageal squamous cell carcinoma (ESCC). We investigated whether alteration of oral microbiota is associated with ESCC risk. Fasting saliva samples were collected from 87 incident and histopathologicallly diagnosed ESCC cases, 63 subjects with dysplasia and 85 healthy controls. All subjects were also interviewed with a questionnaire. V3–V4 region of 16S rRNA was amplified and sequenced by 454-pyrosequencing platform. Carriage of each genus was compared by means of multivariate-adjusted odds ratios derived from logistic regression model. Relative abundance was compared using Metastats method. Beta diversity was estimated using Unifrac and weighted Unifrac distances. Principal coordinate analysis (PCoA) was applied to ordinate dissimilarity matrices. Multinomial logistic regression was used to compare the coordinates between different groups. ESCC subjects had an overall decreased microbial diversity compared to control and dysplasia subjects (P<0.001). Decreased carriage of genera Lautropia, Bulleidia, Catonella, Corynebacterium, Moryella, Peptococcus and Cardiobacterium were found in ESCC subjects compared to non-ESCC subjects. Multinomial logistic regression analyses on PCoA coordinates also revealed that ESCC subjects had significantly different levels for several coordinates compared to non-ESCC subjects. In conclusion, we observed a correlation between altered salivary bacterial microbiota and ESCC risk. The results of our study on the saliva microbiome are of particular interest as it reflects the shift in microbial communities. Further studies are warranted to verify this finding, and if being verified, to explore the underlying mechanisms.  相似文献   

10.
Pulmonary tuberculosis (TB) has gained attention in recent decades because of its rising incidence trend; simultaneously, increasing numbers of studies have identified the relationship between microbiota and chronic infectious diseases. In our work, we enrolled 32 patients with primary TB characterised by unilateral TB lesion formation diagnosed by chest radiographic exam. Bronchoalveolar lavage fluid was taken from both lungs. Twenty-four healthy people were chosen as controls. Pyrosequencing was performed on the V3 hypervariable region of 16S rDNA in all bacterial samples and used as a culture-independent method to describe the phylogenetic composition of the microbiota. Through pyrosequencing, 271,764 amplicons were detected in samples and analysed using tools in the Ribosomal Database Project (RDP) and bioinformatics. These analyses revealed significant differences in the microbiota in the lower respiratory tract (LRT) of TB patients compared with healthy controls; in contrast, the microbiota of intra/extra-TB lesions were similar. These results showed that the dominant bacterial genus in the LRT of TB patients was Cupriavidus and not Streptococcus, which resulted in a significant change in the microbiota in TB patients. The abundance of Mycobacteria and Porphyromonas significantly increased inside TB lesions when compared with non-lesion-containing contralateral lungs. From these data, it can be concluded that Cupriavidus plays an important role in TB’s secondary infection and that in addition to Mycobacteria, Porphyromonas may also be a co-factor in lesion formation. The mechanisms underlying this connection warrant further research.  相似文献   

11.
The pathogenesis of psoriasis, an immune-mediated chronic inflammatory skin disease, remains unclear. Studies have shown an association between psoriasis and intestinal inflammation; in this context, the influence of the gut microbiota on the immune response of psoriasis has become a focus of recent research. The present research evaluated the composition and diversity of the gut microbiota of 21 participants with psoriasis from a Brazilian referral dermatology service compared to 24 healthy controls. A stool sample was collected from each participant at the time of inclusion in the study, and the samples were analysed by sequencing the 16S rRNA gene. The recruitment of research participants involved matching between groups by sex, age, body mass index, comorbidities and smoking and the exclusion of several criteria that could potentially influence the gut microbiota and the interpretation of the data. There was an increase in the Dialister genus and Prevotella copri species in patients with psoriasis compared to the control group. A reduction in the Ruminococcus, Lachnospira and Blautia genera, as well as in the Akkermansia muciniphila species, was also verified in the psoriasis group compared to the control group. Furthermore, patients with psoriasis exhibited less gut microbiota diversity than controls.  相似文献   

12.
The oral cavity serves as another reservoir for gastric Helicobacter pylori and may contribute to the failure of gastric H. pylori eradication therapy. However, changes to the oral microbial composition after gastric H. pylori eradication therapy has not yet been identified. This study aims to dissect whether the oral microbiota is involved and which bacterium mediates the clinic failure in H. pylori eradication. In the present study, the oral microorganisms from patients who had received the gastric H. pylori eradication treatment were analyzed by a high-throughput 16S rRNA deep sequencing. We found that the β diversity and composition of oral microbiota were remarkably changed in the patients who had experienced successful gastric H. pylori eradication treatment (SE group) compared to the failure group (FE group). Significantly enriched families, including Prevotellaceae, Streptococcaceae, Caulobacteraceae, and Lactobacillaceae, were detected in the SE group. In contrast, the bacterial families, such as Weeksellaceae, Neisseriaceae, Peptostreptococcaceae, Spirochaetaceae, and Veillonellaceae, were abundantly expressed in the FE group. Five operational taxonomic units (OTUs) were positively correlated with DOB values, while two OTUs exhibited negative trends. These different enriched OTUs were extensively involved in the 20 metabolic pathways. These results suggest that a balanced environment in the oral microbiota contributes to H. pylori eradication and metabolic homeostasis in humans. Our data demonstrated that the changes in oral microbiota might contribute to the therapeutic effects of antibiotic therapy. Therefore, a different therapy on the detrimental oral microbiota will increase the therapeutic efficacy of antibiotics on H. pylori infection. Open in a separate window  相似文献   

13.
14.
Recurrent Clostridium difficile infection (CDI) can be effectively treated by infusion of a healthy donor faeces suspension. However, it is unclear what factors determine treatment efficacy. By using a phylogenetic microarray platform, we assessed composition, diversity and dynamics of faecal microbiota before, after and during follow-up of the transplantation from a healthy donor to different patients, to elucidate the mechanism of action of faecal infusion. Global composition and network analysis of the microbiota was performed in faecal samples from nine patients with recurrent CDI. Analyses were performed before and after duodenal donor faeces infusion, and during a follow-up of 10 weeks. The microbiota data were compared with that of the healthy donors. All patients successfully recovered. Their intestinal microbiota changed from a low-diversity diseased state, dominated by Proteobacteria and Bacilli, to a more diverse ecosystem resembling that of healthy donors, dominated by Bacteroidetes and Clostridium groups, including butyrate-producing bacteria. We identified specific multi-species networks and signature microbial groups that were either depleted or restored as a result of the treatment. The changes persisted over time. Comprehensive and deep analyses of the microbiota of patients before and after treatment exposed a therapeutic reset from a diseased state towards a healthy profile. The identification of microbial groups that constitute a niche for C. difficile overgrowth, as well as those driving the reinstallation of a healthy intestinal microbiota, could contribute to the development of biomarkers predicting recurrence and treatment outcome, identifying an optimal microbiota composition that could lead to targeted treatment strategies.  相似文献   

15.

Background

The human gastrointestinal tract is inhabited by a very diverse symbiotic microbiota, the composition of which depends on host genetics and the environment. Several studies suggested that the host genetics may influence the composition of gut microbiota but no genes involved in host control were proposed. We investigated the effects of the wild type and mutated alleles of the gene, which encodes the protein called pyrin, one of the regulators of innate immunity, on the composition of gut commensal bacteria. Mutations in MEFV lead to the autoinflammatory disorder, familial Mediterranean fever (FMF, MIM249100), which is characterized by recurrent self-resolving attacks of fever and polyserositis, with no clinical signs of disease in remission.

Methodology/Principal Findings

A total of 19 FMF patients and eight healthy individuals were genotyped for mutations in the MEFV gene and gut bacterial diversity was assessed by sequencing 16S rRNA gene libraries and FISH analysis. These analyses demonstrated significant changes in bacterial community structure in FMF characterized by depletion of total numbers of bacteria, loss of diversity, and major shifts in bacterial populations within the Bacteroidetes, Firmicutes and Proteobacteria phyla in attack. In remission with no clinical signs of disease, bacterial diversity values were comparable with control but still, the bacterial composition was substantially deviant from the norm. Discriminant function analyses of gut bacterial diversity revealed highly specific, well-separated and distinct grouping, which depended on the allele carrier status of the host.

Conclusions/Significance

This is the first report that clearly establishes the link between the host genotype and the corresponding shifts in the gut microbiota (the latter confirmed by two independent techniques). It suggests that the host genetics is a key factor in host-microbe interaction determining a specific profile of commensal microbiota in the human gut.  相似文献   

16.
Despite a long-suspected role in the development of human colorectal cancer (CRC), the composition of gut microbiota in CRC patients has not been adequately described. In this study, fecal bacterial diversity in CRC patients (n=46) and healthy volunteers (n=56) were profiled by 454 pyrosequencing of the V3 region of the 16S ribosomal RNA gene. Both principal component analysis and UniFrac analysis showed structural segregation between the two populations. Forty-eight operational taxonomic units (OTUs) were identified by redundancy analysis as key variables significantly associated with the structural difference. One OTU closely related to Bacteroides fragilis was enriched in the gut microbiota of CRC patients, whereas three OTUs related to Bacteroides vulgatus and Bacteroides uniformis were enriched in that of healthy volunteers. A total of 11 OTUs belonging to the genera Enterococcus, Escherichia/Shigella, Klebsiella, Streptococcus and Peptostreptococcus were significantly more abundant in the gut microbiota of CRC patients, and 5 OTUs belonging to the genus Roseburia and other butyrate-producing bacteria of the family Lachnospiraceae were less abundant. Real-time quantitative PCR further validated the significant reduction of butyrate-producing bacteria in the gut microbiota of CRC patients by measuring the copy numbers of butyryl-coenzyme A CoA transferase genes (Mann–Whitney test, P<0.01). Reduction of butyrate producers and increase of opportunistic pathogens may constitute a major structural imbalance of gut microbiota in CRC patients.  相似文献   

17.
Increasing evidence suggests that perturbations in the intestinal microbiota composition of infants are implicated in the pathogenesis of food allergy (FA), while the actual structure and composition of the intestinal microbiota in human beings with FA remain unclear. Microbial diversity and composition were analyzed with parallel barcoded 454 pyrosequencing targeting the 16S rRNA gene hypervariable V1-V3 regions in the feces of 34 infants with FA (17 IgE mediated and 17 non-IgE mediated) and 45 healthy controls. Here, we showed that several key FA-associated bacterial phylotypes, but not the overall microbiota diversity, significantly changed in infancy fecal microbiota with FA and were associated with the development of FA. The proportion of abundant Bacteroidetes, Proteobacteria, and Actinobacteria phyla were significantly reduced, while the Firmicutes phylum was highly enriched in the FA group (P < 0.05). Abundant Clostridiaceae 1 organisms were prevalent in infants with FA at the family level (P = 0.016). FA-enriched phylotypes negatively correlated with interleukin-10, for example, the genera Enterococcus and Staphylococcus. Despite profound interindividual variability, levels of 20 predominant genera were significantly different between the FA and healthy control groups (P < 0.05). Infants with IgE-mediated FA had increased levels of Clostridium sensu stricto and Anaerobacter and decreased levels of Bacteroides and Clostridium XVIII (P < 0.05). A positive correlation was observed between Clostridium sensu stricto and serum-specific IgE (R = 0.655, P < 0.001). The specific microbiota signature could distinguish infants with IgE-mediated FA from non-IgE-mediated ones. Detailed microbiota analysis of a well-characterized cohort of infants with FA showed that dysbiosis of fecal microbiota with several FA-associated key phylotypes may play a pathogenic role in FA.  相似文献   

18.
Respiratory infections are well-known triggers of chronic respiratory diseases. Recently, culture-independent tools have indicated that lower airway microbiota may contribute to pathophysiologic processes associated with asthma and chronic obstructive pulmonary disease (COPD). However, the relationship between upper airway microbiota and chronic respiratory diseases remains unclear. This study was undertaken to define differences of microbiota in the oropharynx of asthma and COPD patients relative to those in healthy individuals. To account for the qualitative and quantitative diversity of the 16S rRNA gene in the oropharynx, the microbiomes of 18 asthma patients, 17 COPD patients, and 12 normal individuals were assessed using a high-throughput next-generation sequencing analysis. In the 259,572 total sequence reads, α and β diversity measurements and a generalized linear model revealed that the oropharynx microbiota are diverse, but no significant differences were observed between asthma and COPD patients. Pseudomonas spp. of Proteobacteria and Lactobacillus spp. of Firmicutes were highly abundant in asthma and COPD. By contrast, Streptococcus, Veillonella, Prevotella, and Neisseria of Bacteroidetes dominated in the healthy oropharynx. These findings are consistent with previous studies conducted in the lower airways and suggest that oropharyngeal airway microbiota are important for understanding the relationships between the various parts of the respiratory tract with regard to bacterial colonization and comprehensive assessment of asthma and COPD.  相似文献   

19.
Increasing evidence suggests that perturbations in the intestinal microbiota in early infancy are implicated in the pathogenesis of food allergy (FA); existing evidence on the structure and composition of the intestinal microbiota in human beings with FA is limited and conflicting. The main object of the study was to compare the faecal microbiota between healthy and cow’s milk allergy (CMA) infants at the baseline immediately after the diagnosis, and to evaluate the changes in the faecal microbiota after 6?months of treatment of CMA infants with hypoallergenic formula (HF), compared with healthy children fed on standard milk formulae. Sixty infants younger than 4?months of age with challenge-proven CMA and 60 healthy age-matched children were investigated in this prospective case - control follow-up study. Faecal samples were collected at baseline and at 6?months of follow-up, microbial diversity and composition were characterized by high-throughput 16S rRNA sequencing. The average age (±SD) of the infants at inclusion was 2.9?±?1.0?months. Children with CMA have lower gut microbiota diversity and an elevated Enterobacteriaceae to Bacteroidaceae (E/B ratio) in early infancy compared with healthy children (115.8 vs. 0.8, P?=?0.0002). After 6?months of treatment with HF, CMA infants had a higher Lactobacillaceae (6.3% vs. 0.5%, P?=?0.04) and lower Bifidobacteriaceae (0.3% vs. 8.2%, P?=?0.03) and Ruminococcaceae (1.5% vs. 10.5%, P?=?0.03) abundance compared with control children. Conclusion: Low gut microbiota diversity and an elevated E/B ratio in early infancy may contribute to the development of FA, including CMA. A strict elimination diet may weaken FA by reducing E/B ratio and promoting a gut microbiota that would benefit the acquisition of oral tolerance.  相似文献   

20.
Noma (cancrum oris) is a gangrenous disease of unknown etiology affecting the maxillo-facial region of young children in extremely limited resource countries. In an attempt to better understand the microbiological events occurring during this disease, we used phylogenetic and low-density microarrays targeting the 16S rRNA gene to characterize the gingival flora of acute noma and acute necrotizing gingivitis (ANG) lesions, and compared them to healthy control subjects of the same geographical and social background. Our observations raise doubts about Fusobacterium necrophorum, a previously suspected causative agent of noma, as this species was not associated with noma lesions. Various oral pathogens were more abundant in noma lesions, notably Atopobium spp., Prevotella intermedia, Peptostreptococcus spp., Streptococcus pyogenes and Streptococcus anginosus. On the other hand, pathogens associated with periodontal diseases such as Aggregatibacter actinomycetemcomitans, Capnocytophaga spp., Porphyromonas spp. and Fusobacteriales were more abundant in healthy controls. Importantly, the overall loss of bacterial diversity observed in noma samples as well as its homology to that of ANG microbiota supports the hypothesis that ANG might be the immediate step preceding noma.  相似文献   

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