A diagnostic challenge in clinical laboratory: Misidentification of Neisseria subflava as Neisseria meningitidis by MALDI-TOF MS.

MALDI-TOF MS provides fast, easy to perform and cost-effective diagnosis in clinical microbiology laboratories, however in some cases results of MALDI-TOF MS should be confirmed with additional tests. This confirmation is especially important for causes of life-threatening infections like Neisseria meningitidis. In our laboratory, three isolates were identified as N. meningitidis by Bruker MALDI Biotyper (BD, USA) between April 2018 and March 2019 from clinical specimens of blood, sputum, and urine. 16S rRNA sequencing was performed for further investigation. Two of the isolates were identified as Neisseria subflava and only one was confirmed as N. meningitidis by sequencing. These results show that MALDI-TOF MS is not always reliable in the diagnosis of N. meningitidis and clinical microbiologists should confirm these results with additional tests. Also, clinical correlations should be determined. Accurate identification of this microorganism is very important because of the necessity of prophylactic antimicrobial usage and biosafety precautions. Enlarged databases of Neisseria species are needed to overcome this problem.

Neisseria weixii sp. nov., isolated from rectal contents of Tibetan Plateau pika (Ochotona curzoniae).

Three independent isolates (10022T, 10 009 and 10011) of a novel catalase-positive, Gram-stain-negative coccus in the genus Neisseria were obtained from the rectal contents of plateau pika on the Qinghai-Tibet Plateau, PR China. Based on 16S rRNA gene sequence analysis, our newly identified organisms were most closely related to Neisseria iguanae, Neisseria flavescens and Neisseria perflava with similarities ranging from 98.02 to 98.45 %, followed by seven other species in the genus Neisseria. Phylogenetic analysis based on 16S rRNA and rplF genes showed that our three novel isolates group with members of the genus Neisseria. Results of the average nucleotide identity (ANI) analysis confirmed that our isolates are of the same species, and the ANI values between type strain 10022T and other Neisseria species are 74.12-85.06 %, lower than the threshold range of 95-96 %. The major cellular fatty acids for our novel species are C16 : 0 and C16:1ω7c/C16:1ω6c, which along with their phenotypic characteristics can distinguish our isolates from other Neisseria species. On the basis of polyphasic analyses, our isolates are proposed to represent a novel species in genus Neisseria, with the name Neisseria weixii sp. nov. The type strain is 10022T (=DSM 103441T=CGMCC 1.15732T).

Neisseria chenwenguii sp. nov. isolated from the rectal contents of a plateau pika (Ochotona curzoniae).

Two Gram-stain negative, catalase positive, coccus shaped bacteria, designated 10023T and 10010, were isolated from the rectal contents of a plateau pika (Ochotona curzoniae) in Qinghai-Tibet Plateau, China. Based on 16S rRNA gene sequence analysis, phylogenetic trees showed that these two isolates (10023T, 10010) group with members of the genus Neisseria. Additionally, these two isolates exhibited high 16S rRNA gene sequence similarity with Neisseria zalophi CSL 7565T (96.98%), Neisseria wadsworthii WC 05-9715T (96.92%) and Neisseria canis ATCC 14687T (96.79%). Further phylogenetic analysis based on the rplF gene showed that these two novel strains can be easily discriminated from phylogenetically closely related species. Optimal growth was found to occur on BHI agar with 5% defibrinated sheep blood at 37 °C and growth was also observed on nutrient agar, Columbia blood agar and chocolate agar plates; however, growth was not observed on MacConkey agar after 7 days. The major cellular fatty acids of these strains were identified as C16:0 and C16:1ω7c/C16:1ω6c. The complete genome size of the type strain 10023T is 2,496,444 bp, with DNA G+C content of 54.0 mol %. The average nucleotide identity values were 73.5-79.3% between isolate 10023T and reference Neisseria spp. Based on polyphasic analysis, these isolates (10023T and 10010) are considered to represent a novel species in the genus Neisseria, for which the name Neisseria chenwenguii sp. nov. is proposed. The type strain is 10023T (= DSM 103440T = CGMCC 1.15736T).

Use of Andromas and Bruker MALDI-TOF MS in the identification of Neisseria.

Through the past decade, MALDI-TOF MS has been recognized as a fast and robust tool for identification of most bacteria in clinical microbiology. However, the accuracy of this method to identify Neisseria species is still debated, and few data are available about commensal Neisseria species identification. In this study, we assessed two MALDI-TOF MS systems (Bruker Biotyper and Andromas) for the identification of 88, 18, and 29 isolates of Neisseria gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species, respectively. All 88 isolates of N. gonorrhoeae were correctly identified using both systems, and most N. meningitidis and commensal Neisseria species were well identified: only 1/18 isolates of N. meningitidis was misidentified using Bruker Biotyper, and 1 isolate of Neisseria polysaccharea was misidentified as N. meningitidis using both systems. These results strengthen the possibility to use MALDI-TOF MS as a single method for Neisseria identification in routine, with excellent performance for N. gonorrhoeae identification. However, results should be interpreted prudently for N. meningitdis and commensal Neisseria species when isolated from genital and oropharyngeal samples where these both species can coexist.

Genotyping of commensal Neisseria spp strains by pulsed-field gel electrophoresis and 16S rRNA gene sequencing.

BACKGROUND: We investigated the diversity of the primary sequences of the 16S rRNA genes among 46 commensal Neisseria strains and evaluated the use of this approach as a molecular typing tool in comparison with PFGE analysis. METHODS: Identification to the genus was done using conventional methods and API NH (bio-Mérieux® ). Identification to species level was based on 16S rRNA gene sequencing. PFGE analysis was done using SpeI. RESULTS: Fourteen, two, three and fourteen 16S rRNA sequence types were found among twenty Neisseria flavescens, two Neisseria sicca, five Neisseria macacae and nineteen Neisseria mucosa clinical isolates. Forty-three different PFGE patterns were found among the tested strains. CONCLUSION: We demonstrated a high diversity among 16S rRNA genes which was reflected by PFGE analysis.

Neisseria dumasiana sp. nov. from human sputum and a dog's mouth.

Three independent isolates of Gram-reaction-negative cocci collected from two New York State patients and a dog's mouth in California were subjected to a polyphasic analysis. The 16S rRNA gene sequence similarity among these isolates is 99.66 to 99.86 %. The closest species with a validly published name is Neisseria zoodegmatis (98.7 % 16S rRNA gene sequence similarity) with six additional species of the genus Neisseria with greater than 97 % similarity. Average nucleotide identity (ANI) and genome-to-genome distance calculator (GGDC 2.0) analysis on whole genome sequence data support the three novel isolates as being from a single species that is distinct from all other closely related species of the genus Neisseria. Phylogenetic analysis of 16S rRNA gene sequences and ribosomal multilocus sequence typing (rMLST) indicate the novel species belongs in the genus Neisseria. This assignment is further supported by the predominant cellular fatty acids composition of C16 : 0, summed feature 3 (C16 : 1ω7c/C15 : 0iso 2-OH), and C18 : 1ω7c, and phenotypic characters. The name Neisseria dumasiana sp. nov. is proposed, and the type strain is 93087T (=DSM 104677T=LMG 30012 T).

Neisseria arctica sp. nov., isolated from nonviable eggs of greater white-fronted geese (Anser albifrons) in Arctic Alaska.

During the summers of 2013 and 2014, isolates of a novel Gram-stain-negative coccus in the genus Neisseriawere obtained from the contents of nonviable greater white-fronted goose (Anseralbifrons) eggs on the Arctic Coastal Plain of Alaska. We used a polyphasic approach to determine whether these isolates represent a novel species. 16S rRNA gene sequences, 23S rRNA gene sequences, and chaperonin 60 gene sequences suggested that these Alaskan isolates are members of a distinct species that is most closely related to Neisseria canis, Neisseriaanimaloris and Neisseriashayeganii. Analysis of the rplF gene additionally showed that the isolates are unique and most closely related to Neisseriaweaveri. Average nucleotide identity of the whole genome sequence of the type strain was between 71.5 and 74.6 % compared to close relatives, further supporting designation as a novel species. Fatty acid methyl ester analysis showed a predominance of C14 : 0, C16 : 0 and C16 : 1ω7c fatty acids. Finally, biochemical characteristics distinguished the isolates from other species of the genus Neisseria. On the basis of these combined data, the isolates are proposed to represent a novel species of the genus Neisseria, with the name Neisseria arctica sp. nov. The type strain is KH1503T (=ATCC TSD-57T=DSM 103136T).

Neisseria gonorrhoeae Crippled Its Peptidoglycan Fragment Permease To Facilitate Toxic Peptidoglycan Monomer Release.

Neisseria gonorrhoeae (gonococci) and Neisseria meningitidis (meningococci) are human pathogens that cause gonorrhea and meningococcal meningitis, respectively. Both N. gonorrhoeae and N. meningitidis release a number of small peptidoglycan (PG) fragments, including proinflammatory PG monomers, although N. meningitidis releases fewer PG monomers. The PG fragments released by N. gonorrhoeae and N. meningitidis are generated in the periplasm during cell wall remodeling, and a majority of these fragments are transported into the cytoplasm by an inner membrane permease, AmpG; however, a portion of the PG fragments are released into the extracellular environment through unknown mechanisms. We previously reported that the expression of meningococcal ampG in N. gonorrhoeae reduced PG monomer release by gonococci. This finding suggested that the efficiency of AmpG-mediated PG fragment recycling regulates the amount of PG fragments released into the extracellular milieu. We determined that three AmpG residues near the C-terminal end of the protein modulate AmpG's efficiency. We also investigated the association between PG fragment recycling and release in two species of human-associated nonpathogenic Neisseria: N. sicca and N. mucosa Both N. sicca and N. mucosa release lower levels of PG fragments and are more efficient at recycling PG fragments than N. gonorrhoeae Our results suggest that N. gonorrhoeae has evolved to increase the amounts of toxic PG fragments released by reducing its PG recycling efficiency. IMPORTANCE: Neisseria gonorrhoeae and Neisseria meningitidis are human pathogens that cause highly inflammatory diseases, although N. meningitidis is also frequently found as a normal member of the nasopharyngeal microbiota. Nonpathogenic Neisseria, such as N. sicca and N. mucosa, also colonize the nasopharynx without causing disease. Although all four species release peptidoglycan fragments, N. gonorrhoeae is the least efficient at recycling and releases the largest amount of proinflammatory peptidoglycan monomers, partly due to differences in the recycling permease AmpG. Studying the interplay between bacterial physiology (peptidoglycan metabolism) and pathogenesis (release of toxic monomers) leads to an increased understanding of how different bacterial species maintain asymptomatic colonization or cause disease and may contribute to efforts to mitigate disease.

Metagenomics Reveals Dysbiosis and a Potentially Pathogenic N. flavescens Strain in Duodenum of Adult Celiac Patients.

OBJECTIVES: Celiac disease (CD)-associated duodenal dysbiosis has not yet been clearly defined, and the mechanisms by which CD-associated dysbiosis could concur to CD development or exacerbation are unknown. In this study, we analyzed the duodenal microbiome of CD patients. METHODS: The microbiome was evaluated in duodenal biopsy samples of 20 adult patients with active CD, 6 CD patients on a gluten-free diet, and 15 controls by DNA sequencing of 16S ribosomal RNA libraries. Bacterial species were cultured, isolated and identified by mass spectrometry. Isolated bacterial species were used to infect CaCo-2 cells, and to stimulate normal duodenal explants and cultured human and murine dendritic cells (DCs). Inflammatory markers and cytokines were evaluated by immunofluorescence and ELISA, respectively. RESULTS: Proteobacteria was the most abundant and Firmicutes and Actinobacteria the least abundant phyla in the microbiome profiles of active CD patients. Members of the Neisseria genus (Betaproteobacteria class) were significantly more abundant in active CD patients than in the other two groups (P=0.03). Neisseria flavescens (CD-Nf) was the most abundant Neisseria species in active CD duodenum. Whole-genome sequencing of CD-Nf and control-Nf showed genetic diversity of the iron acquisition systems and of some hemoglobin-related genes. CD-Nf was able to escape the lysosomal compartment in CaCo-2 cells and to induce an inflammatory response in DCs and in ex-vivo mucosal explants. CONCLUSIONS: Marked dysbiosis and an abundance of a peculiar CD-Nf strain characterize the duodenal microbiome in active CD patients thus suggesting that the CD-associated microbiota could contribute to the many inflammatory signals in this disorder.

Population and Functional Genomics of Neisseria Revealed with Gene-by-Gene Approaches.

Rapid low-cost whole-genome sequencing (WGS) is revolutionizing microbiology; however, complementary advances in accessible, reproducible, and rapid analysis techniques are required to realize the potential of these data. Here, investigations of the genus Neisseria illustrated the gene-by-gene conceptual approach to the organization and analysis of WGS data. Using the gene and its link to phenotype as a starting point, the BIGSdb database, which powers the PubMLST databases, enables the assembly of large open-access collections of annotated genomes that provide insight into the evolution of the Neisseria, the epidemiology of meningococcal and gonococcal disease, and mechanisms of Neisseria pathogenicity.

Isolation and characterization of Neisseria musculi sp. nov., from the wild house mouse.

Members of the genus Neisseria have been isolated from or detected in a wide range of animals, from non-human primates and felids to a rodent, the guinea pig. By means of selective culture, biochemical testing, Gram staining and PCR screening for the Neisseria-specific internal transcribed spacer region of the rRNA operon, we isolated four strains of the genus Neisseria from the oral cavity of the wild house mouse, Mus musculus subsp. domesticus. The isolates are highly related and form a separate clade in the genus, as judged by tree analyses using either multi-locus sequence typing of ribosomal genes or core genes. One isolate, provisionally named Neisseria musculi sp. nov. (type strain AP2031T=DSM 101846T=CCUG 68283T=LMG 29261T), was studied further. Strain AP2031T/N. musculi grew well in vitro. It was naturally competent, taking up DNA in a DNA uptake sequence and pilT-dependent manner, and was amenable to genetic manipulation. These and other genomic attributes of N. musculi sp. nov. make it an ideal candidate for use in developing a mouse model for studying Neisseria-host interactions.

Neisseria infection of rhesus macaques as a model to study colonization, transmission, persistence, and horizontal gene transfer.

The strict tropism of many pathogens for man hampers the development of animal models that recapitulate important microbe-host interactions. We developed a rhesus macaque model for studying Neisseria-host interactions using Neisseria species indigenous to the animal. We report that Neisseria are common inhabitants of the rhesus macaque. Neisseria isolated from the rhesus macaque recolonize animals after laboratory passage, persist in the animals for at least 72 d, and are transmitted between animals. Neisseria are naturally competent and acquire genetic markers from each other in vivo, in the absence of selection, within 44 d after colonization. Neisseria macacae encodes orthologs of known or presumed virulence factors of human-adapted Neisseria, as well as current or candidate vaccine antigens. We conclude that the rhesus macaque model will allow studies of the molecular mechanisms of Neisseria colonization, transmission, persistence, and horizontal gene transfer. The model can potentially be developed further for preclinical testing of vaccine candidates.

Diversity and homogeneity of oral microbiota in healthy Korean pre-school children using pyrosequencing.

Objectives The purpose of this study was designed to identify the oral microbiota in healthy Korean pre-school children using pyrosequencing. Materials and methods Dental plaque samples were obtained form 10 caries-free pre-school children. The samples were analysed using pyrosequencing. Results The pyrosequencing analysis revealed that, at the phylum level, Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria and Fusobacteria showed high abundance. Also, predominant genera were identified as core microbiome, such as Streptococcus, Neisseria, Capnocytophaga, Haemophilus and Veilonella. Conclusions The diversity and homogeneity was shown in the dental plaque microbiota in healthy Korean pre-school children.

Neisseria weaveri Andersen et al. 1993 is a later heterotypic synonym of Neisseria weaveri Holmes et al. 1993.

Two species of the genus Neisseria, namely Neisseria weaveri Andersen et al. 1993 and Neisseria weaveri Holmes et al. 1993, were simultaneously proposed and described in the same volume of International Journal of Systematic Bacteriology, and have been maintained as heterotypic homonyms. However, the identical 16S rRNA gene sequence and high (99.1 %) average nucleotide identity (ANI) between the genome sequences of the two type strains implied that these two taxa should be united as a single genomic species. To clarify their taxonomic status, phenotypic properties including enzymic activities and substrate-utilization profiles were investigated. The results demonstrated that the two taxa have no pronounced differences and should constitute a single species. Therefore, the reclassification of N. weaveri Andersen et al. 1993 as a later heterotypic synonym of N. weaveri Holmes et al. 1993 is proposed.

Identifying Neisseria species by use of the 50S ribosomal protein L6 (rplF) gene.

The comparison of 16S rRNA gene sequences is widely used to differentiate bacteria; however, this gene can lack resolution among closely related but distinct members of the same genus. This is a problem in clinical situations in those genera, such as Neisseria, where some species are associated with disease while others are not. Here, we identified and validated an alternative genetic target common to all Neisseria species which can be readily sequenced to provide an assay that rapidly and accurately discriminates among members of the genus. Ribosomal multilocus sequence typing (rMLST) using ribosomal protein genes has been shown to unambiguously identify these bacteria. The PubMLST Neisseria database (http://pubmlst.org/neisseria/) was queried to extract the 53 ribosomal protein gene sequences from 44 genomes from diverse species. Phylogenies reconstructed from these genes were examined, and a single 413-bp fragment of the 50S ribosomal protein L6 (rplF) gene was identified which produced a phylogeny that was congruent with the phylogeny reconstructed from concatenated ribosomal protein genes. Primers that enabled the amplification and direct sequencing of the rplF gene fragment were designed to validate the assay in vitro and in silico. Allele sequences were defined for the gene fragment, associated with particular species names, and stored on the PubMLST Neisseria database, providing a curated electronic resource. This approach provides an alternative to 16S rRNA gene sequencing, which can be readily replicated for other organisms for which more resolution is required, and it has potential applications in high-resolution metagenomic studies.

Microbial signature profiles of periodontally healthy and diseased patients.

AIM: To determine microbial profiles that discriminate periodontal health from different forms of periodontal diseases. METHODS: Subgingival biofilm was obtained from patients with periodontal health (27), gingivitis (11), chronic periodontitis (35) and aggressive periodontitis (24), and analysed for the presence of >250 species/phylotypes using HOMIM. Microbial differences among groups were examined by Mann-Whitney U-test. Regression analyses were performed to determine microbial risk indicators of disease. RESULTS: Putative and potential new periodontal pathogens were more prevalent in subjects with periodontal diseases than periodontal health. Detection of Porphyromonas endodontalis/Porphyromonas spp. (OR 9.5 [1.2-73.1]) and Tannerella forsythia (OR 38.2 [3.2-450.6]), and absence of Neisseria polysaccharea (OR 0.004 [0-0.15]) and Prevotella denticola (OR 0.014 [0-0.49], p < 0.05) were risk indicators of periodontal disease. Presence of Aggregatibacter actinomycetemcomitans (OR 29.4 [3.4-176.5]), Cardiobacterium hominis (OR 14.9 [2.3-98.7]), Peptostreptococcaceae sp. (OR 35.9 [2.7-483.9]), P. alactolyticus (OR 31.3 [2.1-477.2]), and absence of Fretibacterium spp. (OR 0.024 [0.002-0.357]), Fusobacterium naviforme/Fusobacterium nucleatum ss vincentii (OR 0.015 [0.001-0.223]), Granulicatella adiacens/Granulicatella elegans (OR 0.013 [0.001-0.233], p < 0.05) were associated with aggressive periodontitis. CONCLUSION: There were specific microbial signatures of the subgingival biofilm that were able to distinguish between microbiomes of periodontal health and diseases. Such profiles may be used to establish risk of disease.

Nasopharyngeal colonization by potentially pathogenic bacteria found in healthy semi-captive wild-born chimpanzees in Uganda.

Information on the chimpanzee nasopharygeal colonization in captive sanctuaries and in the wild is rare. This study was undertaken to establish the nasopharygeal colonization and potential bacterial pathogens in sanctuary chimpanzees as a basis for improving chimpanzee and employee health. Nasopharygeal colonization of 39 healthy chimpanzees were analyzed by microbiological cultivation method and polymerase chain reaction (PCR) targeting the bacterial 16S rRNA gene. We report four major phyla dominated by Proteobacteria (50%), Fermicutes (35.7%), Bacteriodes (7.1%), and Cynobacteria (7.1%) in healthy semi-captive chimpanzees. Further classification based on 7-base oligomers revealed the following genera: Streptococcus, Veillonella, Neisseria, Prevotella, Kingella and unclassified Cynobacteria, Actinobacillus, Bacteriodes and Pasteurellaceae. On microbiological cultivation we were able to identify and characterize some of the bacteria to species level as Klebsiella pneumonie and Pseudomonas aeruginosa being dominant bacteria with 54.7% and 50% colonization, respectively. Of these, Streptococcus, Neisseria, Klebsiella, and Haemophillus have representatives known to potentially cause severe respiratory disease. Our data present important information on chimpanzee nasopharygeal colonization as a guide to understanding disease processes and pharmaceutical therapies required for improving the health of chimpanzees. The results from this study will guide the processes to improve procedures for routine management of sanctuary chimpanzees and use it as a basis for evaluation of future reintroduction possibilities.

Genetic and molecular analyses reveal an evolutionary trajectory for glycan synthesis in a bacterial protein glycosylation system.

Although protein glycosylation systems are becoming widely recognized in bacteria, little is known about the mechanisms and evolutionary forces shaping glycan composition. Species within the genus Neisseria display remarkable glycoform variability associated with their O-linked protein glycosylation (pgl) systems and provide a well developed model system to study these phenomena. By examining the potential influence of two ORFs linked to the core pgl gene locus, we discovered that one of these, previously designated as pglH, encodes a glucosyltransferase that generates unique disaccharide products by using polyprenyl diphosphate-linked monosaccharide substrates. By defining the function of PglH in the glycosylation pathway, we identified a metabolic conflict related to competition for a shared substrate between the opposing glycosyltransferases PglA and PglH. Accordingly, we propose that the presence of a stereotypic, conserved deletion mutation inactivating pglH in strains of Neisseria gonorrhoeae, Neisseria meningitidis, and related commensals, reflects a resolution of this conflict with the consequence of reduced glycan diversity. This model of genetic détente is supported by the characterization of pglH "missense" alleles encoding proteins devoid of activity or reduced in activity such that they cannot exert their effect in the presence of PglA. Thus, glucose-containing glycans appear to be a trait undergoing regression at the genus level. Together, these findings document a role for intrinsic genetic interactions in shaping glycan evolution in protein glycosylation systems.

Comparison of 16S rRNA sequencing with biochemical testing for species-level identification of clinical isolates of Neisseria spp.

We aimed to compare accuracy of genus and species level identification of Neisseria spp. using biochemical testing and 16S rRNA sequence analysis. These methods were evaluated using 85 Neisseria spp. clinical isolates initially identified to the genus level by conventional biochemical tests and API NH system (Bio-Mérieux(®)). In 34 % (29/85), more than one possibility was given by 16S rRNA sequence analysis. In 6 % (5/85), one of the possibilities offered by 16S rRNA gene sequencing, agreed with the result given by biochemical testing. In 4 % (3/85), the same species was given by both methods. 16S rRNA gene sequencing results did not correlate well with biochemical tests.

Oral microbiome sequencing revealed the enrichment of Fusobacterium sp., Porphyromonas sp., Campylobacter sp., and Neisseria sp. on the oral malignant fibroma surface of giant panda.

Introduction: Microbial community composition is closely associated with host disease onset and progression, underscoring the importance of understanding host-microbiota dynamics in various health contexts. Methods: In this study, we utilized full-length 16S rRNA gene sequencing to conduct species-level identification of the microorganisms in the oral cavity of a giant panda (Ailuropoda melanoleuca) with oral malignant fibroma. Results: We observed a significant difference between the microbial community of the tumor side and non-tumor side of the oral cavity of the giant panda, with the latter exhibiting higher microbial diversity. The tumor side was dominated by specific microorganisms, such as Fusobacterium simiae, Porphyromonas sp. feline oral taxon 110, Campylobacter sp. feline oral taxon 100, and Neisseria sp. feline oral taxon 078, that have been reported to be associated with tumorigenic processes and periodontal diseases in other organisms. According to the linear discriminant analysis effect size analysis, more than 9 distinct biomarkers were obtained between the tumor side and non-tumor side samples. Furthermore, the Kyoto Encyclopedia of Genes and Genomes analysis revealed that the oral microbiota of the giant panda was significantly associated with genetic information processing and metabolism, particularly cofactor and vitamin, amino acid, and carbohydrate metabolism. Furthermore, a significant bacterial invasion of epithelial cells was predicted in the tumor side. Discussion: This study provides crucial insights into the association between oral microbiota and oral tumors in giant pandas and offers potential biomarkers that may guide future health assessments and preventive strategies for captive and aging giant pandas.