Prevalence, genetic characteristics, and antimicrobial resistance of staphylococcal isolates from oral cavity and skin surface of healthy individuals in northern Japan.

BACKGROUND: Oral cavity is an ecological niche for colonization of staphylococci, which are a major bacterial species causing community-acquired infections in humans. In this study, prevalence, and characteristics of staphylococci in oral cavity and skin of healthy individuals were investigated in northern Japan. METHODS: Saliva from oral cavity and swab from skin surface of hand were collected and cultured on selective media. Species of the isolates were identified genetically, and ST was determined for S. aureus and S. argenteus. Genes associated with antimicrobial resistance were detected by PCR. RESULTS: Among 166 participants, a total of 75 S. aureus isolates were obtained from 61 individuals (37 %), and recovered more frequently in oral cavity (n = 48) than skin (n = 27). Among 23 STs identified in S. aureus isolates, ST8 (CC8), ST15 (CC15), and ST188 (CC1) were the most common (10 isolates each), with STs of CC1 being dominant (17 isolates). Methicillin-resistant S. aureus (MRSA) was isolated in the skin of two individuals and belonged to ST1 and ST6. Resistance to erythromycin and gentamicin associated with erm(A) and aac(6')-Ie-aph(2")-Ia, respectively, was more commonly found in ST5 and ST8 isolates. One S. argenteus isolate (ST2250, mecA-negative) was recovered from oral cavity of a participant (0.6 %). A total of 186 isolates of coagulase-negative staphylococci (CoNS) were recovered from 102 participants and identified into 14 species, with S. warneri being the most common (n = 52), followed by S. capitis (n = 42), S. saprophyticus (n = 20) and S. haemolyticus (n = 19). mecA was detected in S. saprophyticus, S. haemolyticus, and S. caprae, while arginine-catabolic mobile element (ACME) in only S. capitis and S. epidermidis. CONCLUSION: S. aureus was more prevalent in oral cavity than skin surface, belonging to three major STs, with CC1 being a dominant lineage. The prevalence of antimicrobial resistance was distinct depending on CoNS species.

Real-time NMR analysis of polyhydroxyalkanoate synthase reaction that synthesizes block copolymer comprising glycolate and 3-hydroxybutyrate.

The sequence-regulating polyhydroxyalkanoate (PHA) synthase PhaCAR spontaneously synthesizes the homo-random block copolymer, poly[3-hydroxybutyrate (3HB)]-b-poly[glycolate (GL)-ran-3HB]. In this study, a real-time in vitro chasing system was established using a high-resolution 800 MHz nuclear magnetic resonance (NMR) and 13C-labeled monomers to monitor the polymerization of GL-CoA and 3HB-CoA into this atypical copolymer. Consequently, PhaCAR initially consumed only 3HB-CoA and subsequently consumed both substrates. The structure of the nascent polymer was analyzed by extracting it with deuterated hexafluoro-isopropanol. In the primary reaction product, a 3HB-3HB dyad was detected, and GL-3HB linkages were subsequently formed. According to these results, the P(3HB) homopolymer segment is synthesized prior to the random copolymer segment. This is the first report of its kind which proposes the application of real-time NMR to a PHA synthase assay, paving the way for elucidating the mechanisms of PHA block copolymerization.

Regaining enamel color quality using enamel matrix derivative.

This study aimed to demonstrate and compare the accuracy of tooth shade selection due to the remineralized enamel crystal with enamel matrix derivative (EMD) in vitro. Etched enamel slices were immersed in four types of mineralization buffers for 16 h. Sodium fluoride (NaF) was added to final concentrations of 1-100 ppm with the mineralization buffer that demonstrated the highest mineralization efficiency. EMD was added to the mineralization buffer containing NaF to see if it has any remineralization capacities. The remineralized enamel crystal was analyzed by SEM and XRD. The tooth shade was evaluated by CIE L*a*b*. The results showed that, without NaF, plate-like nanocrystals were formed on the enamel surface, but with NaF, needle-like nanocrystals were formed. By adding EMD, a layer of well-compacted hydroxyapatite crystals was successfully precipitated onto the natural enamel surface. No significant differences were observed in the L* value of the mineralization surface pre-etching and after mineralization buffer containing NaF and EMD. A new method has been developed to recover the color quality of enamel, as well as to mineralize the tooth enamel by constructing hydroxyapatite crystals with mineralization buffers containing NaF and EMD on the etched tooth surface.

Analysis of the cells isolated from epithelial cell rests of Malassez through single-cell limiting dilution.

The epithelial cell rests of Malassez (ERM) are essential in preventing ankylosis between the alveolar bone and the tooth (dentoalveolar ankylosis). Despite extensive research, the mechanism by which ERM cells suppress ankylosis remains uncertain; perhaps its varied population is to reason. Therefore, in this study, eighteen unique clones of ERM (CRUDE) were isolated using the single-cell limiting dilution and designated as ERM 1-18. qRT-PCR, ELISA, and western blot analyses revealed that ERM-2 and -3 had the highest and lowest amelogenin expression, respectively. Mineralization of human periodontal ligament fibroblasts (HPDLF) was reduced in vitro co-culture with CRUDE ERM, ERM-2, and -3 cells, but recovered when an anti-amelogenin antibody was introduced. Transplanted rat molars grown in ERM-2 cell supernatants produced substantially less bone than those cultured in other cell supernatants; inhibition was rescued when an anti-amelogenin antibody was added to the supernatants. Anti-Osterix antibody staining was used to confirm the development of new bones. In addition, next-generation sequencing (NGS) data were analysed to discover genes related to the distinct roles of CRUDE ERM, ERM-2, and ERM-3. According to this study, amelogenin produced by ERM cells helps to prevent dentoalveolar ankylosis and maintain periodontal ligament (PDL) space, depending on their clonal diversity.

Increased integrity of cell-cell junctions accompanied by increased expression of claudin 4 in keratinocytes stimulated with vitamin D3.

The stratified squamous epithelium has a multilayer structure formed by the differentiation of the keratinized epithelium, which covers the skin and oral mucosa. The epithelium plays a central role in regulating the interactions between the immune system and pathogens. The tight junction (TJ) barrier, which is composed of adhesion molecules called claudins (CLDN), is critical for the homeostasis of the skin and oral mucosa. Furthermore, the crucial roles of vitamin D3 (VD3) in the pathogeneses of skin and oral mucosal disease have been suggested. The aim of this in vitro study was to observe the correlations between the integrity of the keratinocyte population and the expression levels of CLDN1 and CLDN4 in gingival epithelial cells, stimulated with VD3. CLDN 1 and 4 expression levels were down and upregulated, respectively, in the cells stimulated with VD3. Additionally, transepithelial electrical resistance (TEER) levels were increased in the stimulated cells when compared to the controls. These findings indicate that CLDN 4 may play a more important role in the TJ barrier than CLDN 1. Hence, the therapeutic effect of VD3 in skin and oral diseases may be regulated by the increase in the expression of CLDN 4.

Biosynthesis of Random-Homo Block Copolymer Poly[Glycolate-ran-3-Hydroxybutyrate (3HB)]-b-Poly(3HB) Using Sequence-Regulating Chimeric Polyhydroxyalkanoate Synthase in Escherichia coli.

Glycolate (GL)-containing polyhydroxyalkanoate (PHA) was synthesized in Escherichia coli expressing the engineered chimeric PHA synthase PhaC AR and coenzyme A transferase. The cells produced poly[GL-co-3-hydroxybutyrate (3HB)] with the supplementation of GL and 3HB, thus demonstrating that PhaC AR is the first known class I PHA synthase that is capable of incorporating GL units. The triad sequence analysis using 1H nuclear magnetic resonance indicated that the obtained polymer was composed of two distinct regions, a P(GL-ran-3HB) random segment and P(3HB) homopolymer segment. The random segment was estimated to contain a 71 mol% GL molar ratio, which was much greater than the value (15 mol%) previously achieved by using PhaC1 P s STQK. Differential scanning calorimetry analysis of the polymer films supported the presence of random copolymer and homopolymer phases. The solvent fractionation of the polymer indicated the presence of a covalent linkage between these segments. Therefore, it was concluded that PhaC AR synthesized a novel random-homo block copolymer, P(GL-ran-3HB)-b-P(3HB).