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Periodontitis affects oral tissues and induces systemic inflammation, which increases the risk of cardiovascular disease and metabolic syndrome. Subgingival plaque accumulation is a trigger of periodontitis. Fusobacterium nucleatum (FN) contributes to subgingival biofilm complexity by intercalating with early and late bacterial colonizers on tooth surfaces. In addition, inflammatory responses to FN are associated with the progression of periodontitis. Nigella sativa Lin. seed, which is known as black cumin (BC), has been used as a herbal medicine to treat ailments such as asthma and infectious diseases. The current study examined the inhibitory effect of BC oil and its active constituents, thymol (TM) and thymoquinone (TQ), on FN‑associated biofilm and inflammation. FN‑containing biofilms were prepared by co‑cultivation with an early dental colonizer, Actinomyces naeslundii (AN). The stability and biomass of FN/AN dual species biofilms were significantly higher compared with FN alone. This effect was retained even with prefixed cells, indicating that FN/AN co‑aggregation is mediated by physicochemical interactions with cell surface molecules. FN/AN biofilm formation was significantly inhibited by 0.1% TM or TQ. Confocal laser scanning microscopy indicated that treatment of preformed FN/AN biofilm with 0.01% of BC, TM or TQ significantly reduced biofilm thickness, and TQ demonstrated a cleansing effect equivalent to that of isopropyl methylphenol. TQ dose‑dependently suppressed TNF‑α production from a human monocytic cell line, THP‑1 exposed to FN, yet showed no toxicity to THP‑1 cells. These results indicated that oral hygiene care using TQ could reduce FN‑associated biofilm and inflammation in gingival tissue.

Otomycosis is a fungal infection of the external auditory canal caused mainly by the genus Aspergillus. Aspergillus luchuensis, an industrially important fungus, is a member of Aspergillus section Nigri. In this report, we present a case of otomycosis due to Aspergillus luchuensis in a 43-year-old female patient. We performed a partial PCR-sequencing of β-tubulin and calmodulin genes to identify the isolate to the species level. Further, we determined the in vitro susceptibility of the isolate to nystatin, clotrimazole and itraconazole according to the Clinical & Laboratory Standards Institute (CLSI) M38-A2 protocol. Accordingly, the minimum inhibitory concentrations of clotrimazole, nystatin and itraconazole were 0.25μg/mL, 0.5μg/mL and 1μg/mL, respectively. This is the first report of clinically relevant isolation of Aspergillus luchuensis identified by a molecular technique as a causative agent of otomycosis.

Disruption of the human gut microbiota by antibiotics can lead to Clostridium difficile (CD)-associated diarrhea. CD overgrowth and elevated CD toxins result in gut inflammation. Herein, we report that a gut symbiont, Bacteroides thetaiotaomicron (BT), suppressed CD toxin production. The suppressive components are present in BT culture supernatant and are both heat- and proteinase K-resistant. Transposon-based mutagenesis indicated that the polysaccharide metabolism of BT is involved in the inhibitory effect. Among the genes identified, we focus on the methylerythritol 4-phosphate pathway gene gcpE, which supplies the isoprenoid backbone to produce the undecaprenyl phosphate lipid carrier that transports oligosaccharides across the membrane. Polysaccharide fractions prepared from the BT culture suppressed CD toxin production in vitro, the inhibitory effect of polysaccharide fractions was reduced in the gcpE mutant (ΔgcpE). The inhibitory effect of BT-derived polysaccharide fraction was abrogated by lysozyme treatment, indicating that cellwall-associated glycans are attributable to the inhibitory effect. BT-derived polysaccharide fraction did not affect CD toxin gene expression or intracellular toxin levels. An autolysis assay showed that CD cell autolysis was suppressed by BT-derived polysaccharide fraction, but the effect was reduced with that of ΔgcpE. These results indicate that cell wall-associated glycans of BT suppress CD toxin release by inhibiting cell autolysis.

Clostridium perfringens possesses the ethanolamine (EA) utilization (eut) system encoded within the eut operon, which utilizes the EA as a carbon, nitrogen and energy source. To determine the role of the eut system in C. perfringens growth, an in-frame deletion of the eutABC genes was made in strain HN13 to generate the eutABC-deleted mutant strain HY1701. Comparison of HN13 and HY1701 growth in media supplemented with 1.0% glucose and/or 1.0% EA showed that glucose enhanced the growth of both strains, whereas EA enhanced HN13 growth, but not that of HY1701, indicating that the eut system is necessary for C. perfringens to utilize EA. The two-component regulatory system EutVW is needed to induce eut gene expression in response to EA whereas the global virulence regulator VirRS differentially controlled eut gene expression depending on glucose and EA availability. To assess the role of the eut system in vivo, an equal number of HN13 and HY1701 cells were injected into the right thigh muscles of mice. Mice infected with HY1701 showed fewer symptoms than those injected with HN13. The mortality rate of mice infected with HY1701 tended to be lower than for mice infected with HN13. In addition, in infected tissues from mice injected with a mixture of HN13 and HY1701, HN13 outnumbered HY1701. PCR screening demonstrated that C. perfringens isolated from gas gangrene and sporadic diarrhea cases carried both eut genes and the perfringolysin O gene (pfoA) as well as the phospholipase C gene (plc). However, pfoA was not detected in isolates from food poisoning patients and healthy volunteers. Culture supernatants prepared from HN13 grown in media containing 7.5% sheep red blood cells induced significantly higher eutB expression levels compared to those from plc- and/or pfoA-deletion mutants. Together, these results indicate that the eut system plays a nutritional role for C. perfringens during histolytic infection.

The human gut commensal Bacteroides fragilis produces sialidases that remove a terminal sialic acid from host-derived polysaccharides. Sialidase is considered to be involved in B. fragilis infection pathology. A native B. fragilis sialidase has been purified and characterized, and was shown to be post-translationally modified by glycosylation. However, the biochemical properties of recombinant B. fragilis sialidase expressed in a heterologous host remain uncharacterized. In this study, we examined the enzymatic properties of the 60-kDa sialidase NanH1 of B. fragilis YCH46, which was prepared as a recombinant protein (rNanH1) in Escherichia coli. In E. coli rNanH1 was expressed as inclusion bodies, which were separated from soluble proteins to allow solubilization of insoluble rNanH1 in a buffer containing 8 M urea and renaturation in refolding buffer containing 100 mM CaCl2 and 50 mM L-arginine. The specific activity of renatured rNanH1 measured using 4-methylumberiferyl-α-D-N-acetyl neuraminic acid as a substrate was 6.16 μmol/min/mg. The optimal pH of rNanH1 ranged from 5.0 to 5.5. The specific activity of rNanH1 was enhanced in the presence of calcium ions. rNanH1 preferentially hydrolyzed the sialyl α2,8 linkage and cleaved sialic acids from mucin and serum proteins (e.g., fetuin and transferrin) but not from α1-acid glycoprotein, which is similar to the previously observed biochemical properties for a native sialidase purified from B. fragilis SBT3182. The results and methods described in this study will be useful for preparing and characterizing recombinant proteins for other B. fragilis sialidase isoenzymes.

Background: Otitis externa is an inflammatory in external auditory canal, with the presentation of otalgia, otorrhea, and pruritus. Bacteria and fungi are the most causative agents of the disease. Although several antifungal and antibacterial agents are usually used to treat it, combination therapy plays an important role in good treatment efficacy. Objectives: According to the problems associated with the treatment of mixed otitis externa, the current study aimed at evaluating the efficacy of ceftazidime powder and topical miconazole (as the case group) versus topical miconazole only (as the control group) to treat mixed otitis externa. Methods: Seventy-two patients with mixed otitis externa were divided into two groups; the case group was treated with ceftazidime powder and topical miconazole, and the control group was treated only with topical miconazole. Both groups were evaluated after two weeks. The diagnosis of mixed otitis externa was based on signs, symptoms, and the presence of bacterial and fungal elements in direct examination and culture. Results: Swelling, itching, and canal discharge were observed in 67.7%, 64.7%, and 90.3% of the patients, respectively in the case group, and 47.1%, 26.3% and 93.1% of the patients, respectively in the control group. Complete resolution of all clinical signs and symptoms occurred in 23 (67.6%) patients in the case group and 11 (28.9%) patients in the control group (P = 0.001). Staphylococcus epidermidis and Pseudomonas aeruginosa were the most common bacteria, and Aspergillus spp. and Candida spp. were the most common fungi identified in the cultures. Conclusions: According to the complete resolution of clinical signs, the application of ceftazidime powder and topical miconazole was better than topical miconazole to treat mixed otitis externa.

Excessive damage to DNA and lipid membranes by reactive oxygen species reduces the viability of bacteria. In the present study, the proliferation of recA‑deficient Escherichia coli strains was revealed to be inhibited by 1% L‑histidine under aerobic conditions. This inhibition of proliferation was not observed under anaerobic conditions, indicating that L‑histidine enhances oxidative DNA damage to E. coli cells. Reverse transcription‑quantitative polymerase chain reaction analysis demonstrated that the expression of recA in E. coli MG1655 increased ~7‑fold following treatment with 10 mM hydrogen peroxide (H2O2) plus 1% L‑histidine, compared with that following exposure to H2O2 alone. L‑histidine increased the genomic fragmentation of E. coli MG1655 following exposure to H2O2. In addition, L‑histidine increased the generation of intracellular hydroxyl radicals in the presence of H2O2 in E. coli cells. Next, our group investigated the disinfection properties of the H2O2 and L‑histidine combination. The combination of 100 mM H2O2 and 1.0% L‑histidine significantly reduced the number of viable cells of extended‑spectrum‑β‑lactamase‑producing E. coli and multidrug‑resistant Pseudomonas aeruginosa, and this treatment was more effective than 100 mM H2O2 alone, but this effect was not observed in methicillin‑resistant Staphylococcus aureus or vancomycin‑resistant Enterococcus faecium. The combination of L‑histidine and H2O2 may be a useful strategy to selectively increase the microbicidal activity of oxidative agents against Gram‑negative bacteria.