Gemella massiliensis sp. nov., a new bacterium isolated from the human sputum.

Thanks to its ability to isolate previously uncultured bacterial species, culturomics has dynamized the study of the human microbiota. A new bacterial species, Gemella massiliensis Marseille-P3249T, was isolated from a sputum sample of a healthy French man. Strain Marseille-P3249T is a facultative anaerobe, catalase-negative, Gram positive, coccus, and unable to sporulate. The major fatty acids were C16:0 (34%), C18:1n9 (28%), C18:0 (15%) and C18:2n6 (13%). Its 16S rRNA sequence exhibits a 98.3% sequence similarity with Gemella bergeri strain 617-93T, its phylogenetically closest species with standing in nomenclature. Its digital DNA-DNA hybridization (dDDH) and OrthoANI values with G. bergeri of only 59.7 ± 5.6% and 94.8%, respectively. These values are lower than the thresholds for species delineation (> 70% and > 95%, respectively). This strain grows optimally at 37 °C and its genome is 1.80 Mbp long with a 30.5 mol% G + C content. Based on these results, we propose the creation of the new species Gemella massilienis sp. nov., strain Marseille-P3249T (= CSUR P3249 = DSMZ 103940).

Polymicrobial abscess following ovariectomy in a mouse.

BACKGROUND: Ovariectomy is a common procedure in laboratory rodents used to create a post-menopausal state. Complications including post-surgical abscess are rarely reported, but merit consideration for the health and safety of experimental animals. CASE PRESENTATION: A female C57/black6 mouse was ovariectomized as part of a cohort study. At Day 14 post-surgery, she developed a visible swelling on the right side, which 7 days later increased in size over 24 h, leading to euthanasia of the animal. Gross pathology was consistent with abscess. A core of necrotic tissue was present in the uterine horn. Abscess fluid and affected tissue were collected for Gram stain and bacteriological culture. The abscess core and fluid yielded three distinct types of bacterial colonies identified by 16S ribosomal RNA sequencing as Streptococcus acidominimus, Pasteurella caecimuris, and a novel species in the genus Gemella. CONCLUSIONS: This is the first report of polymicrobial abscess in a rodent as a complication of ovariectomy, and the first description of a novel Gemella species for which we have proposed the epithet Gemella muriseptica. This presentation represents a potential complication of ovariectomy in laboratory animals.

Purpura fulminans with Lemierre's syndrome caused by Gemella bergeri and Eikenella corrodens: a case report.

BACKGROUND: Gemella bergeri is one of the nine species of the genus Gemella and is relatively difficult to identify. We herein describe the first case of septic shock due to a Gemella bergeri coinfection with Eikenella corrodens. CASE PRESENTATION: A 44-year-old Asian man with a medical history of IgG4-related ophthalmic disease who was prescribed corticosteroids (prednisolone) presented to our hospital with dyspnea. On arrival, he was in shock, and a purpuric eruption was noted on both legs. Contrast enhanced computed tomography showed fluid retention at the right maxillary sinus, left lung ground glass opacity, and bilateral lung irregular opacities without cavitation. Owing to suspected septic shock, fluid resuscitation and a high dose of vasopressors were started. In addition, meropenem, clindamycin, and vancomycin were administered. Repeat computed tomography confirmed left internal jugular and vertebral vein thrombosis. Following this, the patient was diagnosed with Lemierre's syndrome. Furthermore, he went into shock again on day 6 of hospitalization. Additional soft tissue infections were suspected; therefore, bilateral below the knee amputations were performed for source control. Cultures of the exudates from skin lesions and histopathological samples did not identify any pathogens, and histopathological findings showed arterial thrombosis; therefore it was concluded that the second time shock was associated with purpura fulminans. Following this, his general status improved. He was transferred to another hospital for rehabilitation. The blood culture isolates were identified as Gemella bergeri and Eikenella corrodens. Gemella bergeri was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and confirmed by 16S rRNA gene sequencing later. The primary focus of the infection was thought to be in the right maxillary sinus, because the resolution of the fluid retention was confirmed by repeat computed tomography. CONCLUSIONS: Gemella bergeri can be the causative pathogen of septic shock. If this pathogen cannot be identified manually or through commercial phenotypic methods, 16S rRNA gene sequencing should be considered.

The first case report of infective endocarditis caused by Gemella taiwanensis.

Gemella is a facultative anaerobic Gram-positive coccus and a rare cause of infective endocarditis (IE). Gram staining may eventually misidentify the organism, which tends to easily decolorize and manifest as either Gram-negative or Gram-variable. Commercial biochemical tests are often used to identify Gemella, but the methods they employ sometimes lack accuracy. A 52-year-old woman was diagnosed with Gemella taiwanensis IE after initial identification of the pathogen as Gemella haemolysans using biochemical tests combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). She was treated successfully with penicillin, gentamicin, and mitral valve replacement. To our knowledge, this is the first case of IE confirmed by 16S rRNA gene and groEL sequencing to have been caused by G. taiwanensis. The accurate diagnosis of rare or difficult-to-identify pathogens is a major challenge for clinical microbiological laboratories. The concurrent use of molecular methods could lead to the recognition of new or different pathogens.

Composition and Predicted Metabolic Capacity of Upper and Lower Airway Microbiota of Healthy Dogs in Relation to the Fecal Microbiota.

The upper and lower airways of healthy humans are reported to harbor stable and consistent bacterial populations, and the composition of these communities is altered in individuals affected with several respiratory diseases. Data regarding the presence of airway microbiota in other animals are scant and a better understanding of the composition and metabolic function of such bacterial populations is essential for the development of novel therapeutic and diagnostic modalities for use in both veterinary and human medicine. Based on targeted next-generation sequencing of feces and samples collected at multiple levels of the airways from 16 healthy female dogs, we demonstrate that canine airways harbor a topographically continuous microbiota with increasing relative abundance of proteobacterial species from the upper to lower airways. The lung-associated microbiota, as assessed via bronchoalveolar lavage fluid (BALF), was the most consistent between dogs and was dominated by three distinct taxa, two of which were resolved to the species level and one to the level of family. The gene content of the nasal, oropharyngeal, and lung-associated microbiota, predicted using the Phylogenetic Investigations into Communities by Reconstruction of Unobserved States (PICRUSt) software, provided information regarding the glyoxylate and citrate cycle metabolic pathways utilized by these bacterial populations to colonize such nutrient-poor, low-throughput environments. These data generated in healthy subjects provide context for future analysis of diseased canine airways. Moreover, as dogs have similar respiratory anatomy, physiology, and immune systems as humans, are exposed to many of the same environmental stimuli, and spontaneously develop similar respiratory diseases, these data support the use of dogs as a model species for prospective studies of the airway microbiota, with findings translatable to the human condition.

Gemella parahaemolysans sp. nov. and Gemella taiwanensis sp. nov., isolated from human clinical specimens.

Four Gram-staining-positive, catalase-negative, coccoid isolates, designated NTUH_1465(T), NTUH_2196, NTUH_4957 and NTUH_5572(T), were isolated from human specimens. The four isolates displayed more than 99.6% 16S rRNA gene sequence similarity with Gemella haemolysans ATCC 10379(T), and 96.7 to 98.6% similarity with Gemella sanguinis ATCC 700632(T), Gemella morbillorum ATCC 27824(T) or Gemella cuniculi CCUG 42726(T). However, phylogenetic analysis of concatenated sequences of three housekeeping genes, groEL, rpoB and recA, suggested that the four isolates were distinct from G. haemolysans ATCC 10379(T) and other species. Isolates NTUH_2196, NTUH_4957 and NTUH_5572(T) clustered together and formed a stable monophyletic clade. DNA-DNA hybridization values among strains NTUH_1465(T) and NTUH_5572(T) and their phylogenetically related neighbours were all lower than 49%. The four isolates could be distinguished from G. haemolysans and other species by phenotypic characteristics. Based on the phylogenetic and phenotypic results, two novel species Gemella parahaemolysans sp. nov. (type strain NTUH_1465(T) = BCRC 80365(T) = JCM 18067(T)) and Gemella taiwanensis sp. nov. (type strain NTUH_5572(T) = BCRC 80366(T) = JCM 18066(T)) are proposed.

Characterization of oral bacterial diversity of irradiated patients by high-throughput sequencing.

The objective of this study was to investigate the compositional profiles and microbial shifts of oral microbiota during head-and-neck radiotherapy. Bioinformatic analysis based on 16S rRNA gene pyrosequencing was performed to assess the diversity and variation of oral microbiota of irradiated patients. Eight patients with head and neck cancers were involved in this study. For each patient, supragingival plaque samples were collected at seven time points before and during radiotherapy. A total of 147,232 qualified sequences were obtained through pyrosequencing and bioinformatic analysis, representing 3,460 species level operational taxonomic units (OTUs) and 140 genus level taxa. Temporal variations were observed across different time points and supported by cluster analysis based on weighted UniFrac metrics. Moreover, the low evenness of oral microbial communities in relative abundance was revealed by Lorenz curves. This study contributed to a better understanding of the detailed characterization of oral bacterial diversity of irradiated patients.

Bacterial diversity in the saliva of patients with different oral hygiene indexes.

The objective of the present study was to evaluate the bacterial diversity in the saliva of patients with different oral hygiene indexes using of two 16S rRNA gene libraries. Each library was composed of samples from patients with different averages of the differentiated Silness-Löe biofilm index: the first library (A) with an index between 1.0 and 3.0 (considered a high index) and the second library (B) between 0 and 0.5 (considered a low index). Saliva DNA was extracted and the 16S rRNA gene was amplified and cloned. The obtained sequences were compared with those stored at NCBI and RDP GenBank. The saliva of patients with high index presented five known genera - Streptococcus, Granulicatella, Gemella, Veillonella and Peptostreptococcus - and 33.3% of nonculturable bacteria grouped into 23 operational taxonomic units (OTUs). The saliva of patients with low index differed significantly from the first library (p=0.000) and was composed of 42 OTUs distributed into 11 known genera - Streptococcus, Granulicatella, Gemella, Veillonella, Oribacterium, Haemophilus, Escherichia, Neisseria, Prevotella, Capnocytophaga, Actinomyces - including 24.87% of nonculturable bacteria. It was possible to conclude that there is greater bacterial diversity in the saliva of patients with low dental plaque in relation to patients with high dental plaque.

Microbial profiles in saliva from children with and without caries in mixed dentition.

OBJECTIVE: The purpose of this study was to determine the bacterial profiles in saliva of the isolated children for studying caries etiology. MATERIALS AND METHODS: Samples were collected from isolated children from 6 to 8years old including 20 caries-free (dmfs=0) (healthy) and 30 caries-active individuals (dmfs>8) (patients). 16S rRNA genes were amplified by PCR from bacterial DNA of saliva sample and labeled via incorporation of Cy3-dCTP in second nested PCR. After hybridization of labeled amplicons on HOMIM, the microarray slides were scanned and original data acquired from professional software. RESULTS: Collectively, 94 bacterial species or clusters representing six bacterial phyla and 30 genera were detected. A higher bacterial diversity was observed in patients than in healthy samples. Statistical analyses revealed eight species or clusters were detected more frequently in diseased patients than in healthy samples, while six different species were detected more frequently in healthy as compared to diseased patients. CONCLUSION: The diversity of microbe within saliva derived from isolated population increased in caries-active status, and there are some bacteria in salivary flora can be as candidate biomarkers for caries prognosis in mixed dentition. The imbalances in the resident microflora may be the ultimate mechanism of dental caries.

The subgingival microbiota of Papillon-Lefèvre syndrome.

BACKGROUND: There is little information about the microbiologic profiles of periodontal lesions in Papillon-Lefèvre syndrome (PLS) and the significance of bacteria in the pathogenesis of periodontitis in these patients. This comprehensive analysis of the subgingival microbiota in patients with PLS used 16S ribosomal RNA (rRNA) clonal analysis and the 16S rRNA-based Human Oral Microbe Identification Microarray (HOMIM). METHODS: Thirteen patients with PLS from seven unrelated families volunteered for this microbiologic study. Subgingival plaque was collected with sterile paper points from multiple sites with ≥5 mm probing depth, and whole genomic DNA was extracted. The 16S rRNA genes were amplified, cloned, and sequenced. The samples were then probed for ≈300 predominant oral bacterial species using the HOMIM. RESULTS: The most commonly detected phylotypes in the clonal analysis were Gemella morbillorum, Gemella haemolysans, Granulicatella adiacens, Lachnospiraceae OT 100 (EI074), Parvimonas micra, Selenomonas noxia, and Veillonella parvula. As a group, streptococci were commonly detected in these individuals. In the HOMIM analysis, a total of 170 bacterial species/phylotypes were detected, with a range of 40 to 80 species per patient with PLS. Of these, 12 bacterial species were detected in medium to high levels in ≥50% of the individuals. The high-frequency strains were clustered into eight groups: Aggregatibacter actinomycetemcomitans, Campylobacter spp., Capnocytophaga granulosa, G. morbillorum, P. micra, Porphyromonas endodontalis, Streptococcus spp., and Tannerella forsythia. CONCLUSIONS: The subgingival microbiota in PLS is diverse. Periodontal pathogens commonly associated with chronic and aggressive periodontitis and opportunistic pathogens may be associated with the development of severe periodontitis in patients with PLS.

Bacterial diversity in the saliva of patients with different oral hygiene indexes

The objective of the present study was to evaluate the bacterial diversity in the saliva of patients with different oral hygiene indexes using of two 16S rRNA gene libraries. Each library was composed of samples from patients with different averages of the differentiated Silness-Löe biofilm index: the first library (A) with an index between 1.0 and 3.0 (considered a high index) and the second library (B) between 0 and 0.5 (considered a low index). Saliva DNA was extracted and the 16S rRNA gene was amplified and cloned. The obtained sequences were compared with those stored at NCBI and RDP GenBank. The saliva of patients with high index presented five known genera - Streptococcus, Granulicatella, Gemella, Veillonella and Peptostreptococcus - and 33.3% of nonculturable bacteria grouped into 23 operational taxonomic units (OTUs). The saliva of patients with low index differed significantly from the first library (p=0.000) and was composed of 42 OTUs distributed into 11 known genera - Streptococcus, Granulicatella, Gemella, Veillonella, Oribacterium, Haemophilus, Escherichia, Neisseria, Prevotella, Capnocytophaga, Actinomyces - including 24.87% of nonculturable bacteria. It was possible to conclude that there is greater bacterial diversity in the saliva of patients with low dental plaque in relation to patients with high dental plaque.

O objetivo do presente estudo foi avaliar a diversidade bacteriana da saliva de pacientes com diferentes índices de higiene bucal através da construção de duas bibliotecas do gene 16S rRNA. Cada biblioteca foi composta por amostras de saliva de pacientes com índice de biofilme dental de Silness-Löe diferenciado, sendo a primeira (A) com índice de 1,0 a 3,0 (denominada de alto índice) e a segunda (B), entre 0 a 0,5 (denominada de baixo índice). O DNA da saliva foi extraído e o gene 16S rRNA foi amplificado, clonado e sequenciado. As sequências obtidas foram comparadas com aquelas armazenadas no GenBank do NCBI e RDP. A saliva de pacientes com alto índice de biofilme dental apresentou cinco gêneros conhecidos: Streptococcus, Granulicatella, Gemella, Veillonella e Peptostreptococcus e 33,3% de bactérias não-cultivadas, agrupados em 23 unidades taxonômicas operacionais (UTOs). A saliva de pacientes com baixo índice de biofilme dental, foi diferente significativamente da primeira (p=0,000) e foi composta de 42 UTOs, distribuídas em 11 gêneros conhecidos: Streptococcus, Granulicatella, Gemella, Veillonella, Oribacterium, Haemophilus, Escherichia, Neisseria, Prevotella, Capnocytophaga, Actinomyces, além de 24,87% de bactérias não-cultivadas. Pode-se concluir que existe maior diversidade bacteriana na saliva de pacientes com baixo índice de biofilme dental em relação a pacientes com alto índice de biofilme dental.

Use of groESL as a target for identification of Abiotrophia, Granulicatella, and Gemella species.

We determined the groESL sequences of three species of nutritionally variant streptococci (Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans) and three Gemella species (Gemella morbillorum, Gemella haemolysans, and Gemella sanguinis). The nucleotide sequence similarities between the groES and groEL genes of the above genera were 41.7 to 85.9% and 63.7 to 84.3%, respectively. The intraspecies similarities of groESL sequences for the isolates of Abiotrophia and Granulicatella species were 94.4 to 97.8% for groES and 94.0 to 98.2% for groEL. For Ge. morbillorum and Ge. sanguinis, all strains showed the same groESL spacer length (8 bp), and sequence identities within species were >97.8% for groES and >96.1% for groEL. However, higher intraspecies heterogeneity was observed in Ge. haemolysans. Phylogenetic analysis of groEL sequences separated the 6 isolates of Ge. haemolysans into two subgroups. Among these isolates, three isolates with the same groESL spacer region length (45 bp) clustered together but were distant from the ATCC reference strain (with a spacer length of 8 bp). The remaining three isolates, with a spacer length of 50 or 8 bp, clustered together. Although 16S rRNA gene sequence analysis did not provide enough discrimination for the 6 Ge. haemolysans isolates, rpoB gene sequence analysis supported the subgrouping. Based on the obtained groESL sequences, we developed a multiplex PCR that enables simple, rapid, and accurate identification of Abiotrophia, Granulicatella, and Gemella at the genus level. This assay would be helpful for identifying these fastidious and slow-growing organisms in clinical laboratories.