A Case of Scrofuloderma in a Patient on JAK Inhibitor Treatment for Rheumatoid Arthritis.

A 78-year-old woman with rheumatoid arthritis, who was started on baricitinib five or six months earlier, was referred to our hospital due to a subcutaneous abscess in her right axilla. Contrast-enhanced chest, abdomen, and pelvis computed tomography showed subcutaneous abscesses in her right axilla and lymphadenopathy with calcification. Cultures from the subcutaneous abscess and skin biopsy specimens were positive for Mycobacterium tuberculosis. These findings led to the diagnosis of scrofuloderma associated with tuberculous lymphadenitis. She was started on an antitubercular regimen of isoniazid, rifampicin, pyrazinamide, and ethambutol as the initial phase treatment (first 2 months), followed by isoniazid and rifampicin for 4 months (total 6 months). After 6 months of antitubercular treatment, the abscesses and lymphadenitis disappeared. Although cases of tuberculosis during JAK inhibitor treatment are rare, they are serious adverse events that require caution.

Antimicrobial effects of epigallocatechin-3-gallate, a catechin abundant in green tea, on periodontal disease-associated bacteria.

OBJECTIVE: Epigallocatechin-3-gallate (EGCG), a catechin abundant in green tea, exhibits antibacterial activity. In this study, the antimicrobial effects of EGCG on periodontal disease-associated bacteria (Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Fusobacterium nucleatum, and Fusobacterium periodontium) were evaluated and compared with its effects on Streptococcus mutans, a caries-associated bacterium. RESULTS: Treatment with 2 mg/ml EGCG for 4 h killed all periodontal disease-associated bacteria, whereas it only reduced the viable count of S. mutans by about 40 %. Regarding growth, the periodontal disease-associated bacteria were more susceptible to EGCG than S. mutans, based on the growth inhibition ring test. As for metabolism, the 50 % inhibitory concentration (IC50) of EGCG for bacterial metabolic activity was lower for periodontal disease-associated bacteria (0.32-0.65 mg/ml) than for S. mutans (1.14 mg/ml). Furthermore, these IC50 values were negatively correlated with the growth inhibition ring (r = -0.73 to -0.86). EGCG induced bacterial aggregation at the following concentrations: P. gingivalis (>0.125 mg/ml), F. periodonticum (>0.5 mg/ml), F. nucleatum (>1 mg/ml), and P. nigrescens (>2 mg/ml). S. mutans aggregated at an EGCG concentration of > 1 mg/ml. CONCLUSION: EGCG may help to prevent periodontal disease by killing bacteria, inhibiting bacterial growth by suppressing bacterial metabolic activity, and removing bacteria through aggregation.

Lactic Acid Bacteria in the Human Oral Cavity: Assessing Metabolic Functions Relevant to Oral Health and Disease.

Many perceive lactic acid bacteria as beneficial for health. They are recognized for preventing abnormal fermentation and spoilage of ingested foods by producing lactic acid, which aids in gut acidification. Moreover, lactic acid bacteria are extensively employed in food science. In contrast, lactic acid bacteria in the oral cavity are often perceived as pathogenic factors contributing to the development of dental caries. As a consequence, substantial research has been conducted in oral and dental sciences to explore lactic acid bacteria and the oral microbiome. This research primarily involves analyzing bacterial flora, investigating metabolic activities such as acid production, and investigating metabolic regulation within the oral environment. The oral cavity serves as the gateway to the digestive tract and respiratory system, characterized by a constantly fluctuating environment that significantly impacts the metabolic activity of lactic acid bacteria. Hence, when investigating oral lactic acid bacteria, it is crucial to adopt research plans and methodologies that account for these metabolic environment changes. In this section, we present some of the methods employed in our study.

Effects of Nitrate and Nitrite on Plaque pH Decrease and Nitrite-Producing and -Degrading Activities of Plaque in vitro.

INTRODUCTION: The purpose of this study was to investigate the effects of nitrate and nitrite on the pH-lowering activity of human plaque, the nitrite-producing and -degrading activities of human plaque, and their correlation. METHODS: Nitrate and nitrite were added to human plaque suspensions collected from the buccal aspect of maxillary molars of patients visiting a general dental clinic, and changes in pH were measured with and without glucose addition. Nitrite-producing and -degrading activities were evaluated by adding nitrate and nitrite to the plaque suspension and measuring the increase and decrease in nitrite with Griess reagent, respectively. RESULTS: The addition of nitrate inhibited both endogenous and glucose-induced plaque pH-lowering. The addition of glucose enhanced the production of nitrite from nitrate by about 3.3-fold. The addition of nitrite also inhibited endogenous plaque pH-lowering, but the addition of glucose promoted nitrite degradation by only about 1.1-fold. Nitrite-producing activity was positively correlated with age, but not with nitrite-degrading activity. CONCLUSION: This study revealed that nitrite was produced from nitrate and inhibited the pH-lowering activity of human plaque, which may contribute to caries control. Both nitrite-producing and -degrading activities occurred in human plaque, but no correlation was found between them. Furthermore, nitrite production was enhanced by glucose metabolism, which may function as a self-regulatory mechanism (resilience) to prevent excessive acidification by glucose metabolism.

Molecular microbiological profiling of bottled unsweetened tea beverages: A screening experiment.

To explore the potential storage and safety of drinking leftover bottled tea beverages from various manufacturers after direct drinking from bottles, we conducted a screening experiment on the growth of salivary bacteria in plastic bottles of tea. The diluted saliva samples from 10 participants were inoculated into the test bottled beverages, which resulted in bacteria, particularly former members of the genus Lactobacillus, growing in some green tea beverages with a neutral pH. In contrast, tea beverages with less bacterial growth contained Streptococcus spp., and the leftovers may be safe to store and drink again.

Characteristics of Visual Cognition in Patients with Anoxic Encephalopathy: An Eye-tracking Study.

Objectives: : Despite the frequent occurrence of visual cognitive impairment after anoxic encephalopathy, only a few studies have analyzed gaze movements following encephalopathy. This study determined the visual cognitive characteristics of patients with anoxic encephalopathy using an eye-tracking system. Methods: : This study included ten patients with anoxic encephalopathy and ten age/sex-matched controls. Factors for anoxic encephalopathy onset and brain imaging findings were extracted from medical records. An eye-tracking system was used to track eye movements during three visual search tasks (pop-out, serial search, and saliency) in patient and healthy control groups. The average target search time, number of saccades, and number of fixations to salient stimuli were compared. Results: : Stagnant blood flow, observed in six of ten patients, was the most common cause of disease onset, four of whom exhibited hypoperfusion in bilateral occipital or parietal lobes on single-photon emission computed tomography. The patient group required longer search times during all visual search tasks and a higher number of saccades during pop-out and serial search tasks. However, no significant difference was observed between the two groups for the number of fixations to salient stimuli during the saliency task. Conclusions: : Following anoxic encephalopathy, bottom-up (pop-out task) and top-down (serial search task) gaze control were considered impaired because of extensive parieto-occipital lobe damage after blood flow stagnation. Patients exhibited reduced top-down function for finding targets (serial search task) but relatively retain inhibitory function for salient stimuli (saliency task). Gaze analysis can be used to reveal the clinical characteristics of anoxic encephalopathy.

A Case of Mirror-Image Crossed Thalamic Aphasia With Jargon Agraphia.

In right-handed individuals, aphasia resulting from right hemisphere damage is termed crossed aphasia and has a very low occurrence rate. Additionally, aphasia due to thalamic lesions often involves hemorrhage, with infarction cases less frequently reported. We present the case of an 81-year-old right-handed female who developed aphasia due to a right thalamic infarction. She exhibited characteristics typical of thalamic aphasia observed in left thalamic lesions. Furthermore, jargon agraphia manifested during writing tasks. This may suggest disinhibition of the left hemisphere writing motor memory by the right hemisphere language function.

Noninvasive graft monitoring using donor-derived cell-free DNA in Japanese liver transplantation.

AIM: To evaluate the use of donor-derived cell-free DNA (dd-cfDNA) in diagnosing graft injuries in Japanese liver transplantation (LTx), including family-related living donors. METHODS: A total of 321 samples from 10 newly operated LTx recipients were collected to monitor the early dynamics of dd-cfDNA levels after LTx. Fifty-five samples from 55 recipients were collected during protocol biopsies (PB), whereas 36 samples from 27 recipients were collected during event biopsies, consisting of 11 biopsy-proven acute rejection (AR), 20 acute dysfunctions without rejection (ADWR), and 5 chronic rejections. The levels of dd-cfDNA were quantified using a next-generation sequencer based on single nucleotide polymorphisms. RESULTS: The dd-cfDNA levels were elevated significantly after LTx, followed by a rapid decline to the baseline in patients without graft injury within 30 days post-LTx. The dd-cfDNA levels were significantly higher in the 11 samples obtained during AR than those obtained during PB (p < 0.0001), which decreased promptly after treatment. The receiver operator characteristic curve analysis of diagnostic ability yielded areas under the curve of 0.975 and 0.897 for AR (rejection activity index [RAI] ≥3) versus PB and versus non-AR (ADWR + PB). The dd-cfDNA levels during AR were elevated earlier and correlated more strongly with the RAI (r = 0.740) than aspartate aminotransferase/alanine aminotransferase. The dd-cfDNA levels were neither associated with graft fibrosis based on histology nor the status of donor-specific antibodies in PB samples. CONCLUSIONS: Donor-derived cell-free DNA serves as a sensitive biomarker for detecting graft injuries in LTx. Further large-scale cohort studies are warranted to optimize its use in differentiating various post-LTx etiologies.

Application of omics technologies in cariology research: A critical review with bibliometric analysis.

OBJECTIVES: To review the application of omics technologies in the field of cariology research and provide critical insights into the emerging opportunities and challenges. DATA & SOURCES: Publications on the application of omics technologies in cariology research up to December 2022 were sourced from online databases, including PubMed, Web of Science and Scopus. Two independent reviewers assessed the relevance of the publications to the objective of this review. STUDY SELECTION: Studies that employed omics technologies to investigate dental caries were selected from the initial pool of identified publications. A total of 922 publications with one or more omics technologies adopted were included for comprehensive bibliographic analysis. (Meta)genomics (676/922, 73 %) is the predominant omics technology applied for cariology research in the included studies. Other applied omics technologies are metabolomics (108/922, 12 %), proteomics (105/922, 11 %), and transcriptomics (76/922, 8 %). CONCLUSION: This study identified an emerging trend in the application of multiple omics technologies in cariology research. Omics technologies possess significant potential in developing strategies for the detection, staging evaluation, risk assessment, prevention, and management of dental caries. Despite the numerous challenges that lie ahead, the integration of multi-omics data obtained from individual biological samples, in conjunction with artificial intelligence technology, may offer potential avenues for further exploration in caries research. CLINICAL SIGNIFICANCE: This review presented a comprehensive overview of the application of omics technologies in cariology research and discussed the advantages and challenges of using these methods to detect, assess, predict, prevent, and treat dental caries. It contributes to steering research for improved understanding of dental caries and advancing clinical translation of cariology research outcomes.

Historical Cohort Study of Congenital Isolated Hypoganglionosis of the Intestine: Determining the Best Surgical Interventions.

No standard diagnostic method or surgical treatment for congenital isolated hypoganglionosis (CIHG) has been established. This study aimed to analyze the clinical outcomes of patients with CIHG and identify the best surgical interventions provided thus far. Data on surgical interventions in 19 patients were collected between 1992 and 2020, including the type of enterostomy, type of revision, and length of the intestines. Ganglion cells in the myenteric plexus were enumerated using Hu C/D staining. The ratio of the length of the small intestine to its height was defined as the intestinal ratio (IR). The outcomes were assessed using the stoma output, growth parameters including the body mass index (BMI), and parenteral nutrition (PN) dependency. All patients required a diverting enterostomy. The IR ranged from 0.51 to 1.75 after multiple non-transplant surgeries. The stoma types were tube-stoma, end-stoma, Santulli-type, and Bishop-Koop (BK)-type. Patients with Santulli- or BK-type stomas had better BMIs and less PN dependency in terms of volume than those with end-stomas or tube-stomas. Two patients with BK-type stomas were off PN, and three who underwent an intestinal transplantation (Itx) achieved enteral autonomy. The management of CIHG involves a precise diagnosis using Hu C/D staining, neonatal enterostomy, and stoma revision using the adjusted IR and Itx if other treatments do not enable enteral autonomy.

Green Tea-Derived Catechins Suppress the Acid Productions of Streptococcus mutans and Enhance the Efficiency of Fluoride.

Green tea-derived catechins, which can be divided into galloylated (epicatechin gallate: ECG, epigallocatechin gallate: EGCG) and non-galloylated (catechin: C, epicatechin: EC, epigallocatechin: EGC) catechins, are considered to be the main contributors to the caries control potential of green tea. In this study, we intended to compare the antimicrobial effects of these representative green tea-derived catechins and their combined effects with fluoride on the acid production and aggregation of Streptococcus mutans. The effects of different catechins on the growth, aggregation and acid production of S. mutans, and the combined effect of catechins and potassium fluoride (2 mm at pH 7.0, 0.3 mm at pH 5.5) on S. mutans acid production were measured by anaerobic culture, turbidity changes due to aggregation, and pH-stat methods. Molecular docking simulations were also performed to investigate the interactions between catechins and membrane-embedded enzyme II complex (EIIC), a component of the phosphoenolpyruvate-dependent phosphotransferase system (sugar uptake-related enzyme). ECG or EGCG at 1 mg/mL significantly inhibited the growth of S. mutans, induced bacterial aggregation, and decreased glucose-induced acid production (p < 0.05). All catechins were able to bind to EIIC in silico, in the following order of affinity: EGCG, ECG, EGC, EC, and C. Furthermore, they enhanced the inhibitory effects of fluoride at pH 5.5 and significantly inhibited S. mutans acid production by 47.5-86.6% (p < 0.05). These results suggest that both galloylated and non-galloylated catechins exhibit antimicrobial activity, although the former type demonstrates stronger activity, and that the caries control effects of green tea may be due to the combined effects of multiple components, such as catechins and fluoride. The detailed mechanisms underlying these phenomena and the in vivo effect need to be explored further.

Abnormal distribution of fat tissue and its association with intestinal failure-associated liver disease in children and adolescents with long-time parenteral nutrition support: A case-control study.

BACKGROUND: Patients with intestinal failure (IF) often present with abnormal body composition characterized by high fat mass. However, the distribution of fat and its association with the development of IF-associated liver disease (IFALD) remain unclear. This study aims to investigate the body composition and its relationship with IFALD in older children and adolescents with IF. METHODS: This retrospective case-control study enrolled patients with IF receiving parenteral nutrition (PN) at Keio University Hospital who initiated PN before the age of 20 years (cases). The control group included patients with abdominal pain, with available computed tomography (CT) scan and anthropometric data. CT scan images of the third lumbar vertebra (L3) were used for body composition analysis and compared between the groups. Liver histology was compared with CT scan findings in IF patients who underwent biopsy. RESULTS: Nineteen IF patients and 124 control patients were included. To account for age distribution, 51 control patients were selected. The median skeletal muscle index was 33.9 (29.1-37.3) in the IF group and 42.1 (39.1-45.7) in the control group (P < 0.01). The median visceral adipose tissue index (VATI) was 9.6 (4.9-21.0) in the IF group and 4.6 (3.0-8.3) in the control group (P = 0.018). Among the 13 patients with IF who underwent liver biopsies, 11 (84.6%) had steatosis, and there was a tendency for fibrosis to correlate with VATI. CONCLUSION: Patients with IF exhibit low skeletal muscle mass and high visceral fat, which may be related to liver fibrosis. Routine monitoring of body composition is recommended.

Chapter 11: Future Perspectives in the Study of Dental Caries.

Dental caries is a disease that affects people of all ages since demineralization and remineralization of tooth surfaces occur in everyone's mouths, and caries lesions develop when there is an imbalance between demineralization and remineralization. In this way, teeth are exposed to a risk of caries. Prevention strategies aiming at "zero caries" and treatments aiming at "tooth recovery and regeneration" are the two main areas of caries research, and both basic and clinical research are required in these fields. The following future perspectives of caries research were identified: The disease concept of caries is undergoing rapid structural changes, as it will increasingly become a disease of all generations: Changes in our understanding of caries etiology (from cariogenic pathogens in the specific plaque hypothesis to the oral microbiome in the ecological plaque hypothesis) will alter the concept of caries-associated bacteria (from mutans streptococci to a group of bacteria with high acid-producing capacity and acid-tolerance or acidogenic/aciduric bacteria). In the field of prevention, more individualized, site-specific, and high-precision examinations for risk assessment and diagnostic methods, including genetic tests, will be developed, and advanced preventive, curative, and regenerative treatments will become possible. To achieve this, interdisciplinary, multidisciplinary, and transdisciplinary research are essential, and collaboration and fusion with other sciences, such as material science, engineering, food science, and nutritional science, are required. Furthermore, in order to put the results of such research into practice in society, it will be necessary to promote industry-academia collaborations; promote behavioral change through sociological approaches; and correct economic, informational, and educational inequalities. The sociological approach requires the coupling of epidemiology and data science as well as the validation of clinical applications, and artificial intelligence will play a powerful role in such analyses.

Potential benefit of rapid genetic testing for Pallister-Hall syndrome.

Pallister-Hall syndrome (PHS) is defined as a group of characteristic manifestations caused by a monoallelic GLI3 pathogenic variant. A two-month-old infant was referred to our institution because of undetermined sex. The infant had atypical genitalia with postaxial polysyndactyly, a hypothalamic mass, and an imperforate anus. We identified a known pathogenic variant of the GLI3 gene within one week and diagnosed the infant with PHS. The parents assigned the infant as male, considering the 46,XY karyotype, normal testosterone secretion, possible male identity, and the natural history of PHS. In infants with atypical genitalia and other malformations, such as polydactyly, a hypothalamic mass, or an imperforate anus, rapid GLI3 testing may provide information for planning lifelong management, including sex assignment.

Questionnaire survey on the satisfaction of SimEx dental education system.

Background/purpose: The SimEx is dental training system that applies new technology in a computerized dental simulator. The purpose of this study was to understand the usability satisfaction of the SimEx dental education and evaluation system by dental students and dentists at Tohoku University. Materials and methods: In this study, the Tohoku University IRB execution number was 2020-3-33. The number of subjects accepted was 59 at Tohoku University and divided into 4 groups based on years of clinical experience (Group A: 0 years; Group B: 1-2 years; Group C: 2-5 years; Group D: at least 5 years), and a total of 58 usability questionnaires were collected. Subjects completed the SimEx Usability Satisfaction Questionnaire after operating the SimEx (EPED Inc., Kaohsiung, Taiwan) course, which contained 16 questions. Results: Among the 58 questionnaires collected by Tohoku University, there were 19 undergraduate students (4th∼6th grade), 12 post-graduate students, 14 residents, and 13 dentists. Significant differences between Group A and Group B, and between Group A and Group D were found (P < 0.05). The same results were obtained for the "experience satisfaction index". In the items where significant differences were found, longer clinical experience tended to result in lower scores. Conclusion: From these results, we can conclude that the SimEx education and evaluation system facilitates students' self-learning, and this system is very useful for continued study and clinical skill training for dentists, especially for students and junior dentists with high usability satisfaction.

In situ detection of endogenous proteolytic activity and the effect of inhibitors on tooth root surface.

OBJECTIVES: The aim of this study was to clarify the distribution and activity of proteolytic enzymes and examine the inhibitory effects of various substances on this proteolytic activity on tooth root surfaces in situ. METHODS: Disk-shaped bovine tooth root samples were partly pretreated in acid solution (50 mM lactic acid buffer, pH 4.0) for 48 h. The fluorescence intensity of samples was detected with fluorescent substrate solution for collagenase and gelatinase using a stereoscopic fluorescence microscope for 60 min. The acid-pretreated and non-acid-pretreated root samples were treated with chlorhexidine (CHX), sodium fluoride (NaF), epigallocatechin gallate (EGCG), and calcium hydroxide (Ca(OH)2) for 10 min, and silver diamine fluoride (SDF) for 10, 30, and 60 s. These samples were immersed in the fluorescence substrate solution at pH 7.0, and the fluorescence intensity of samples was detected. The fluorescence intensity was calculated using analysis software. RESULTS: Gelatinase activity was detected in root samples. Gelatinase activity of the acid-pretreated side was significantly higher than that of the non-acid-pretreated side (1.63 times) at 60 min. CHX, EGCG, Ca(OH)2, and SDF decreased the gelatinase activity of root samples, while NaF had no effect. CONCLUSIONS: Gelatinase activity was detected from the root in situ and it was increased by acid-pretreatment. CHX, EGCG, Ca(OH)2, and SDF inhibited gelatinase activity. CLINICAL SIGNIFICANCE: Substances that inhibit proteolytic activity found in this research method will be useful for root caries prevention.

Glycolysis-Related Gene Analyses Indicate That DEPDC1 Promotes the Malignant Progression of Oral Squamous Cell Carcinoma via the WNT/ß-Catenin Signaling Pathway.

Increasing evidence suggests that aerobic glycolysis is related to the progression of oral squamous cell carcinoma (OSCC). Hence, we focused on glycolysis-related gene sets to screen for potential therapeutic targets for OSCC. The expression profiles of OSCC samples and normal controls were obtained from The Cancer Genome Atlas (TCGA). Then, the differentially expressed gene sets were selected from the official GSEA website following extraction of the differentially expressed core genes (DECGs). Subsequently, we tried to build a risk model on the basis of DECGs to predict the prognosis of OSCC patients via Cox regression analysis. Furthermore, crucial glycolysis-related genes were selected to explore their biological roles in OSCC. Two active glycolysis-related pathways were acquired and 66 DECGs were identified. Univariate Cox regression analysis showed that six genes, including HMMR, STC2, DDIT4, DEPDC1, SLC16A3, and AURKA, might be potential prognostic factors. Subsequently, a risk formula consisting of DEPDC1, DDIT4, and SLC16A3 was established on basis of the six molecules. Furthermore, DEPDC1 was proven to be related to advanced stage cancer and lymph node metastasis. Moreover, functional experiments suggested that DEPDC1 promoted the aerobic glycolysis, migration, and invasion of OSCC via the WNT/ß-catenin pathway. The risk score according to glycolysis-related gene expression might be an independent prognostic factor in OSCC. In addition, DEPDC1 was identified as playing a carcinogenic role in OSCC progression, suggesting that DEPDC1 might be a novel biomarker and therapeutic target for OSCC.

Combined Treatment with Fluoride and Antimicrobial Peptide GH12 Efficiently Controls Caries in vitro and in vivo.

Combining fluoride and antimicrobial agents enhances regulation of acid and exopolysaccharide production by biofilms. The combination also weakens the acidogenic and aciduric bacteria that contribute to caries, achieving stronger caries-controlling effects with lower concentrations of fluoride. In previous studies, antimicrobial peptide GH12 has been shown to inhibit lactic acid and exopolysaccharide synthesis in various cariogenic biofilm models, and reduce the proportion of acidogenic bacteria and Keyes caries scores in a rat caries model. The current study aimed to elucidate the effect of a combination of low concentrations of sodium fluoride (NaF) and GH12 and to determine the mechanism by which GH12/NaF combination controls caries. The GH12/NaF combination contained 8 mg/L GH12 and 250 ppm NaF. A rat caries model was built, and rat dental plaque was sampled and cultivated on bovine enamel slabs in vitro and subjected to short-term treatment (5 min, 3 times/day). The caries-controlling effects were evaluated using Keyes scoring and transverse microradiography. The results showed that the GH12/NaF combination significantly decreased the onset and development of dental caries, as well as mineral content loss and lesion depth in vitro (p < 0.05). For the caries-controlling mechanisms, 16S rRNA sequencing of in vivo dental plaque revealed that populations of commensal bacteria Rothia spp. and Streptococcus parasanguinis increased in the GH12/NaF group. In contrast, Veillonella, Lactobacillus, and Streptococcus mutans decreased. Furthermore, the GH12/NaF combination significantly reduced biomass, lactic acid, and exopolysaccharides production of in vitro biofilm (p < 0.05). Overall, fluoride and GH12 efficiently arrested caries development and demineralization by regulating the microbiota and suppressing acid and exopolysaccharide production in biofilms.

Investigation of drug resistance of caries-related streptococci to antimicrobial peptide GH12.

Dental caries is associated with caries-related streptococci and antimicrobial agents have been widely used for caries control, but troubled by antibiotic resistance. This study aimed to investigate the intrinsic and acquired resistance of caries-related streptococci to antimicrobial peptide GH12, which was proven promising for caries control, and preliminarily explore the phenotypic changes and whole genome of stable acquired resistant strains. In this study, susceptibility assays and resistance assays were performed, followed by stability assays of resistance, to evaluate the intrinsic resistance and the potential resistance of caries-related streptococci. Then, the phenotypic changes of the stable acquired resistant strain were explored. The whole genome of the resistant strain was sequenced and analyzed by second-generation and third-generation high-throughput sequencing technologies. Streptococcus gordonii and Streptococcus sanguinis were intrinsically resistant to GH12 compared to cariogenic Streptococcus mutans. Acquired GH12 resistance in one S. sanguinis and four S. mutans clinical strains was transient but stable in one S. mutans strain (COCC33-14). However, acquired resistance to daptomycin (DAP) and chlorhexidine in all strains was stable. Furthermore, the COCC33-14 showed cross-resistance to DAP and delayed growth rates and a lower population. However, no drug-resistant gene mutation was detected in this strain, but 6 new and 5 missing genes were found. Among them, annotation of one new gene (gene 1782|COCC33-14R) is related to the integral component of the membrane, and one missing gene rpsN is associated with the metabolism and growth of bacteria. The results indicate that stable resistant mutants of caries-related streptococci could hardly be selected by exposure to consecutive sublethal GH12, but the risk still existed. Resistance in COCC33-14R is mainly related to changes in the cell envelope.

Profiling of the microbiota of breast milk before and after feeding with an artificial nipple.

OBJECTIVES: Breast milk is a valuable and useful source of nutrition; however, surplus milk is routinely discarded for hygiene reasons despite an unclear scientific basis. Here, we profiled the microbiota of expressed breast milk before and after feeding with an artificial nipple and examined the bacterial survival in breast milk stored at 4 °C. METHODS: Eleven mother-baby pairs were included in the study. Samples of expressed breast milk were collected before and after feeding with an artificial nipple and examined both immediately (0 h) and after storage for 3 and 12 h at 4 °C. Each sample was inoculated onto a blood agar plate and incubated anaerobically and aerobically at 37 °C. Genomic DNA was extracted from individual bacterial colonies, which were identified by 16S rRNA gene sequencing. RESULTS: Before feeding, the bacterial counts at 0 and 12 h were (1.4 ± 1.6) × 105 colony-forming units (CFU)/mL and (1.4 ± 0.6) × 105 CFU/mL, respectively. Staphylococcus (47.7% and 41.9%, respectively), Cutibacterium (20.7% and 36.0%, respectively), and Streptococcus (16.1% and 6.6%, respectively) were identified among the samples. In contrast, after feeding, the bacterial counts at 0 and 12 h were (2.7 ± 1.7) × 105 CFU/mL and (2.1 ± 2.5) × 105 CFU/mL, respectively. Staphylococcus (30.1% and 37.4%, respectively), Cutibacterium (11.7% and 31.7%, respectively), and Streptococcus (41.5% and 25.2%, respectively), were identified among the samples. CONCLUSIONS: Bacteria were present in the breast milk before feeding. Although the main component of the microbiota shifted from Staphylococcus to Streptococcus species after feeding, these results suggest that surplus expressed breast milk may be preserved safely in a refrigerator for at least 12 h after feeding with an artificial nipple.