Phylogenomic and molecular markers based studies on Staphylococcaceae and Gemella species. Proposals for an emended family Staphylococcaceae and three new families (Abyssicoccaceae fam. nov., Salinicoccaceae fam. nov. and Gemellaceae fam. nov.) harboring four new genera, Lacicoccus gen. nov., Macrococcoides gen. nov., Gemelliphila gen. nov., and Phocicoccus gen. nov.

The family Staphylococcacae and genus Gemella contain several organisms of clinical or biotechnological importance. We report here comprehensive phylogenomic and comparative analyses on 112 available genomes from species in these taxa to clarify their evolutionary relationships and classification. In a phylogenomic tree based on 678 core proteins, Gemella species were separated from Staphylococcacae by a long branch indicating that they constitute a distinct family (Gemellaceae fam. nov.). In this tree, Staphylococcacae species formed two main clades, one encompassing the genera Aliicoccus, Jeotgalicoccus, Nosocomiicoccus and Salinicoccus (Family "Salinicoccaceae"), while the other clade consisted of the genera Macrococcus, Mammaliicoccus and Staphylococcus (Family Staphylococcaceae emend.). In this tree, species from the genera Gemella, Jeotgalicoccus, Macrococcus and Salinicoccus each formed two distinct clades. Two species clades for these genera are also observed in 16S rRNA gene trees and supported by average amino acid identity analysis. We also report here detailed analyses on protein sequences from Staphylococcaceae and Gemella genomes to identify conserved signature indels (CSIs) which are specific for different genus and family-level clades. These analyses have identified 120 novel CSIs robustly demarcating different proposed families and genera. The identified CSIs provide independent evidence that the genera Gemella, Jeotgalicoccus, Macrococcus and Salinicoccus consist of two distinct clades, which can be reliably distinguished based on multiple exclusively shared CSIs. We are proposing transfers of the species from the novel clades of the above four genera into the genera Gemelliphila gen. nov., Phocicoccus gen. nov., Macrococcoides gen. nov. and Lacicoccus gen. nov., respectively. The identified CSIs also provide strong evidence for division of Staphylococcaceae into an emended family Staphylococcaceae and two new families, Abyssicoccaceae fam. nov. and Salinicoccaceae fam. nov. All of these families can be reliably demarcated based on several exclusively shared CSIs.

The First Case of Infectious Spondylitis Caused by Gemella bergeri.

Gemella bergeri, a member of the genus Gemella, is a facultatively anaerobic, Gram-positive cocci. G. bergeri is a component of normal oral flora; however, it can become pathogenic and cause infections in patients with poor oral hygiene. A 78-year-old man was admitted to a hospital with a complaint of increasing posterior neck pain and lower back pain for 2 weeks. MRI was suggestive of infectious spondylitis at the C3-C4 level with prevertebral abscess formation, anterior epidural abscess formation. We identified Gemella bergeri in closed pus obtained during the surgery. Herein, we describe the first case of infective spondylitis caused by G. bergeri.

Mediation effect of intestinal microbiota on the relationship between fiber intake and colorectal cancer.

Higher fiber intake has been associated with a lower risk of colorectal cancer (CRC) and has been shown to protect against CRC based on probable evidence. Recent studies revealed a possible mechanism whereby the interaction between intestinal microbiota and fiber intake mediates CRC risk. However, the specific intestinal bacteria and the amount of these bacteria involved in this mechanism are not fully known. Therefore, this single-center study aimed to determine whether specific intestinal bacteria mediated the relationship between fiber intake and CRC risk. We enrolled patients who received colonoscopy at National Cancer Center Hospital. This cross-sectional study included 180 patients with clinically diagnosed CRC and 242 controls. We conducted a causal mediation analysis to assess the natural indirect effect and natural direct effect of specific intestinal bacteria on association between fiber intake and CRC risk. The median age was 64 (interquartile range, 54-70) years, and 58% of the participants were males. We used metagenomics for profiling gut microbiomes. The relative abundance of each species in each sample was calculated. Among the candidate, Fusobacterium nucleatum and Gemella morbillorum had a significant natural indirect effect based on their highest fiber intake compared to the lowest fiber intake, with a risk difference (95% confidence interval, proportion of mediation effect) of -0.06 [-0.09 to -0.03, 23%] and -0.03 [-0.06 to -0.01, 10.5%], respectively. Other bacteria did not display natural indirect effects. In conclusion, Fusobacterium nucleatum and Gemella morbillorum were found to mediate the relationship between fiber intake and CRC risk.

Gemella morbillorum mitral valve endocarditis in a patient with a history of mitral valve annuloplasty.

A woman with a history of congenital heart disease status post multiple valve operations including mitral valve repair presented with 2 months of low back pain and general malaise. Blood cultures returned positive for Gram-positive cocci. While transthoracic echocardiography did not identify vegetations, transoesophageal echocardiography visualised vegetations on the patient's mitral valve, which had previously undergone repair with annuloplasty. The patient was found to have infectious endocarditis (IE), caused by Gemella morbillorum The patient was treated with over 6 weeks of intravenous antibiotics. Cases of Gemella-associated IE are rare and largely relegated to case reports. This report aims to contribute to the literature regarding this subject, and to further characterise the presentation and treatment of Gemella-associated IE. Additionally, this report emphasises the importance of maintaining a high suspicion of IE in a patient with non-specific malaise in the setting of prior cardiac valve operation.

Comparative susceptibility of Gemella morbillorum to 13 antimicrobial agents.

The in vitro activity of 13 antimicrobials against clinical isolates of Gemella morbillorum showed good susceptibility to clindamycin, all beta-lactams agents studied except cefoxitin (MIC90, 4 µg/ml) and fluoroquinolones. There was 36% metronidazole resistance.

Contemporary experience of Abiotrophia, Granulicatella and Gemella bacteremia.

BACKGROUND: Abiotrophia, Granulicatella, and Gemella are gastrointestinal microbiota, gram-positive cocci that behave like viridans group streptococci. Despite the low incidence of bacteremia from these organisms, they can lead to infective endocarditis (IE) and other clinical syndromes. Due to scant data, we aim to describe detailed clinical features, management, and outcomes of patients with bacteremia from these organisms. METHODS: We reviewed all adult patients who developed Abiotrophia, Granulicatella, or Gemella bacteremia from 2011 to 2020, at Mayo Clinic. RESULTS: We identified 238 patients with positive blood culture for these organisms. Of those, 161 (67.6%) patients were deemed to have bacteremia of clinical significance; 62 (38.5%) were neutropenic, - none of whom were diagnosed with IE. The primary source of bacteremia for the neutropenic group was the gastrointestinal tract. Among 161 patients, echocardiography was obtained in 88 (54.7%) patients, especially those with unknown sources of bacteremia. A total of 19 cases had IE: 5 (26.3%) Abiotrophia, 11 (57.9%) Granulicatella, and 3 (15.8%) Gemella. Based on known IE scoring systems, the negative predictive value at established cutoffs for these scores, performed with our cohort were 95.9%, 100% and 97.9% for NOVA, HANDOC and DENOVA scores, respectively. We also found that the penicillin-non-susceptible rate was high in Abiotrophia (66.7%) and Granulicatella (53.7%). CONCLUSIONS: We described unique characteristics of Abiotrophia, Granulicatella, and Gemella bacteremia at our institution. Clinical significance, clinical syndrome, their proclivity of endocarditis, and susceptibility pattern should be thoroughly reviewed when encountering these organisms.

Time- and Species-Dependent Bacterial Adhesion to Titanium over Short Exposure Periods: An In Vitro Study.

A considerable percentage of dental implant patients experience biofilm-mediated peri-implant disease following transmucosal abutment application. Bacterial adhesion is an early step in biofilm development. Our purpose was to assess adhesion of specific bacterial species to titanium over short exposure periods. Eight bacterial species were selected for this analysis: Streptococcus oralis, Streptococcus mitis, Gemella haemolysans, Streptococcus gordonii, Streptococcus sanguinis, Neisseria flavescens, Streptococcus salivarius, and Pseudomonas aeruginosa. We cultured each species with appropriate media and exposed titanium foil discs to the bacteria for 60, 15, 5, 1, or 0.25 minutes. Optical density at 600-nm wavelength (OD600) was assessed for the baseline inoculum and each species/exposure combination. The proportion of bacteria adherent to titanium was determined for each experimental condition. Striking titanium adhesion was noted for all evaluated species even when exposure time was limited to 15 seconds. Strategies to limit bacterial adhesion at dental implant surfaces may offer potential for improved treatment outcomes and preservation of peri-implant health.

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).

Multi-valvular infective endocarditis from Gemella morbillorum.

Gemella morbillorum is increasingly implicated in infectious endocarditis. Our patient presented with anaemia and renal failure with evidence of infarcts and embolic disease. He was found to have endocarditis with an organism that could not speciate with standard culture methods requiring matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) for identification and susceptibilities. While involvement of mitral and aortic valves can be expected with Gemella, he had rare involvement of the pulmonic valve in a structurally normal heart. Although bacteriological cure was achieved, due to the locally destructive nature of Gemella, he ultimately required valve replacements for heart failure resolution. Workup for commonly implicated pathologies associated with G. morbillorum led to suspicion of gastrointestinal malignancy with findings of occult bleeding prompting an ongoing evaluation. With improved access to advanced diagnostics, G. morbillorum has been increasingly identified in infectious endocarditis. Given its destructive nature, it is important for clinicians to consider this organism is difficult to identify isolates.

Gemella haemolysans inhibits the growth of the periodontal pathogen Porphyromonas gingivalis.

The oral microbiome plays an important role in the human microbial community and in maintaining the health of an individual. Imbalances in the oral microbiome may contribute to oral and systemic diseases. The progression of periodontal disease is closely related to the growth of bacteria, such as Porphyromonas gingivalis, in the oral cavity. However, the pathogen growth mechanism specific to periodontal disease remains unknown. This study aimed to identify bacteria associated with periodontal health by focusing on hemolytic bacteria. Unstimulated saliva samples were collected from ten periodontitis patients and five healthy subjects to detect and identify the presence of hemolytic bacteria. The saliva of healthy subjects contained a higher proportion of G. haemolysans than saliva samples from patients with periodontitis. Growth inhibition assays indicated that the protein components contained in the culture supernatant of G. haemolysans directly suppressed the growth of P. gingivalis. This study shows that the presence of G. haemolysans in saliva is associated with periodontal health and that it inhibits the growth of P. gingivalis in vitro.

Subgingival microbiome of deep and shallow periodontal sites in patients with rheumatoid arthritis: a pilot study.

BACKGROUND: Subgingival microbiome in disease-associated subgingival sites is known to be dysbiotic and significantly altered. In patients with rheumatoid arthritis (RA), the extent of dysbiosis in disease- and health-associated subgingival sites is not clear. METHODS: 8 RA and 10 non-RA subjects were recruited for this pilot study. All subjects received full oral examination and underwent collection of subgingival plaque samples from both shallow (periodontal health-associated, probing depth ≤ 3mm) and deep subgingival sites (periodontal disease-associated, probing depth ≥ 4 mm). RA subjects also had rheumatological evaluation. Plaque community profiles were analyzed using 16 S rRNA sequencing. RESULTS: The phylogenetic diversity of microbial communities in both RA and non-RA controls was significantly higher in deep subgingival sites compared to shallow sites (p = 0.022), and the overall subgingival microbiome clustered primarily according to probing depth (i.e. shallow versus deep sites), and not separated by RA status. While a large number of differentially abundant taxa and gene functions was observed between deep and shallow sites as expected in non-RA controls, we found very few differentially abundant taxa and gene functions between deep and shallow sites in RA subjects. In addition, compared to non-RA controls, the UniFrac distances between deep and shallow sites in RA subjects were smaller, suggesting increased similarity between deep and shallow subgingival microbiome in RA. Streptococcus parasanguinis and Actinomyces meyeri were overabundant in RA subjects, while Gemella morbillorum, Kingella denitrificans, Prevotella melaninogenica and Leptotrichia spp. were more abundant in non-RA subjects. CONCLUSIONS: The aggregate subgingival microbiome was not significantly different between individuals with and without rheumatoid arthritis. Although the differences in the overall subgingival microbiome was driven primarily by probing depth, in contrast to the substantial microbiome differences typically seen between deep and shallow sites in non-RA patients, the microbiome of deep and shallow sites in RA patients were more similar to each other. These results suggest that factors associated with RA may modulate the ecology of subgingival microbiome and its relationship to periodontal disease, the basis of which remains unknown but warrants further investigation.

Bacterial resistance to minocycline after adjunctive minocycline microspheres during periodontal maintenance: A randomized clinical trial.

BACKGROUND: Despite widespread use, the impact of minocycline hydrochloride microspheres on the shifts of oral bacterial species resistant to minocycline remains unknown. This study aimed at examining the percentage and taxonomy of minocycline-resistant isolates in saliva and subgingival plaque samples before and after minocycline microspheres application in periodontitis patients during maintenance. METHODS: Patients received supra- and sub-gingival debridement with (test) or without (control) minocycline microspheres application to sites with probing depth >4 mm and were clinically monitored at baseline, 1, 3, and 6 months. Samples were collected at baseline, 1 and 6 months and analyzed via cultivation with or without 4 µg/mL minocycline. Percentage of resistant strains was determined by colony counting and taxonomy by checkerboard DNA-DNA hybridization. Significant clinical changes were sought with the Mann-Whitney test and differences in percentage of resistant isolates with the Friedman and Mann-Whitney tests. RESULTS: Groups showed similar clinical improvements. Mean percentage of resistant isolates rose at 1 month and decreased at 6 months in saliva and plaque samples in test group (P <0.05) but remained unchanged in control group. Percentage of resistant isolates of Gemella morbillorum and Eubacterium saburreum increased significantly at 6 months in both groups. Antibiotic resistance by Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Porphyromonas gingivalis was either absent or infrequent. CONCLUSION: Minocycline microspheres result in transient selection of minocycline resistant species in saliva and subgingival plaque samples.

Identification of Culturable and Nonculturable Microorganisms, Lipopolysaccharides, and Lipoteichoic Acids From Root Canals of Teeth With Endodontic Failure.

AIM: To elucidate the presence of apical periodontitis in the root canal of teeth with secondary/persistent infection, including composition of microbiota, levels of endotoxins and lipoteichoic acid (LTA), and clinical implications of these findings. METHOD: Samples were collected from root canals of 50 patients who needed endodontic retreatment and had radiographic evidence of apical periodontitis. Microorganisms were identified by using the culture technique and biochemical tests. Nested-polymerase chain reaction (nested-PCR) was used to identify 17 species of specific bacteria. Lipopolysaccharides (LPS) and LTAs were quantified by using, respectively, limulus amebocyte lysate and enzyme-linked immunosorbent assay tests. RESULTS: Bacteria were detected in all samples by culture and molecular methods. A total of 154 gram-positive strains, of 188 strains isolated, were found in the root canals by culture. Enterococcus faecalis and Gemella morbillorum were the most prevalent species identified by the biochemical tests, whereas molecular analyses (nested-PCR) showed a high frequency of P. gingivalis, E. faecalis, and Fusobacterium nucleatum. LPS and LTA were detected in all samples, with mean values being 3.52 EU/mL and 597.83 pg/mL, respectively. Significant statistical correlations were found between levels of LTA and clinical features. CONCLUSION: Despite the prevalence of gram-positives, the microbiota present in secondary/persistent infections showed a large variety of species. Within this diversity, associations were found between specific bacteria and clinical features. In addition, higher levels of LTA were statistically associated with larger periapical radiolucent areas, but no correlation between this feature and LPS was found.

Association of exacerbation phenotype with the sputum microbiome in chronic obstructive pulmonary disease patients during the clinically stable state.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a progressive, life-threatening lung disease with increasing prevalence and incidence worldwide. Increasing evidence suggests that lung microbiomes might play a physiological role in acute exacerbations of COPD. The objective of this study was to characterize the association of the microbiota and exacerbation risk or airflow limitation in stable COPD patients. METHODS: The sputum microbiota from 78 COPD outpatients during periods of clinical stability was investigated using 16S rRNA V3-V4 amplicon sequencing. The microbiome profiles were compared between patients with different risks of exacerbation, i.e., the low risk exacerbator (LRE) or high risk exacerbator (HRE) groups, and with different airflow limitation severity, i.e., mild to moderate (FEV1 ≥ 50; PFT I) or severe to very severe (FEV1 < 50; PFT II). RESULTS: The bacterial diversity (Chao1 and observed OTUs) was significantly decreased in the HRE group compared to that in the LRE group. The top 3 dominant phyla in sputum were Firmicutes, Actinobacteria, and Proteobacteria, which were similar in the HRE and LRE groups. At the genus level, compared to that in the LRE group (41.24%), the proportion of Streptococcus was slightly decreased in the HRE group (28.68%) (p = 0.007). However, the bacterial diversity and the proportion of dominant bacteria at the phylum and genus levels were similar between the PFT I and PFT II groups. Furthermore, the relative abundances of Gemella morbillorum, Prevotella histicola, and Streptococcus gordonii were decreased in the HRE group compared to those in the LRE group according to linear discriminant analysis effect size (LEfSe). Microbiome network analysis suggested altered bacterial cooperative regulation in different exacerbation phenotypes. The proportions of Proteobacteria and Neisseria were negatively correlated with the FEV1/FVC value. According to functional prediction of sputum bacterial communities through Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis, genes involved in lipopolysaccharide biosynthesis and energy metabolism were enriched in the HRE group. CONCLUSION: The present study revealed that the sputum microbiome changed in COPD patients with different risks of exacerbation. Additionally, the bacterial cooperative networks were altered in the HRE patients and may contribute to disease exacerbation. Our results provide evidence that sputum microbiome community dysbiosis is associated with different COPD phenotypes, and we hope that by understanding the lung microbiome, a potentially modifiable clinical factor, further targets for improved COPD therapies during the clinically stable state may be elucidated.

The role of oral microbiome in pemphigus vulgaris.

While the impact of oral microbiome dysbiosis on autoimmune diseases has been partially investigated, its role on bullous diseases like Pemphigus Vulgaris (PV) is a totally unexplored field. This study aims to present the composition and relative abundance of microbial communities in both healthy individuals and patients with oral PV lesions. Ion Torrent was used to apply deep sequencing of the bacterial 16S rRNA gene to oral smear samples of 15 healthy subjects and 15 patients. The results showed that the most dominant phyla were Firmicutes (55.88% controls-c vs 61.27% patients-p, p value = 0.002), Proteobacteria (9.17%c vs 12.33%p, p value = 0.007) and Fusobacteria (3.39%c vs 4.09%p, p value = 0.03). Alpha diversity showed a significant difference in the number of genera between patients and controls (p value = 0.04). Beta diversity showed statistical differences in the microbial community composition between two groups. Fusobacterium nucleatum, Gemella haemolysans and Parvimonas micra were statistically abundant in patients. We noticed the characteristic fetor coming out of oral PV lesions. Most of anaerobic bacteria responsible for oral halitosis are periopathogenic. Though, only F. nucleatum and P. micra were differentially abundant in our patients. Especially, F. nucleatum has been reported many times as responsible for bad breath. Furthermore, Streptococcus salivarius and Rothia mucilaginosa, species mostly associated with clean breath, were found in relative abundance in the healthy group. Consequently, the distinct malodor observed in PV patients might be attributed either to the abundance of F. nucleatum and P. micra and/or to the lower levels of S. salivarius and R. mucilanginosa in oral lesions.

Metagenomic Analyses Expand Bacterial and Functional Profiling Biomarkers for Colorectal Cancer in a Hainan Cohort, China.

This study was conducted for the metagenomic analysis of stool samples from CRC affected individuals to identify biomarkers for CRC in Hainan, the only tropical island province of China. The gut microbiota of CRC patients differed significantly from that of healthy and reference database cohorts based on Aitchison distance and Bray-Cutis distance but there was no significant difference in alpha diversity. Furthermore, at the species level, 68 species were significantly altered including 37 CRC-enriched, such as, Fusobacterium nucleatum, Parvimonas micra, Gemella morbillorum, Citrobacter portucalensis, Alloprevotella sp., Shigella sonnei, Coriobacteriaceae bacterium, etc. Sixty-seven different metabolic pathways were acquired, and pathways involved in the synthesis of many amino acids were significantly declined. Besides, 2 identified antibiotic resistance genes performed well (area under the receive-operation curve AUC = 0.833, 95% CI 58.51-100%) compared with virulence factor genes. The results of the present study provide region-specific bacterial and functional biomarkers of gut microbiota for CRC patients in Hainan. Microbiota is considered as a non-invasive biomarker for the detection of CRC. Gut microbiota of different geographic regions should be further studied to expand the understanding of markers, especially for the China cohort due to diverse nationalities and lifestyles.