Molecular Characterization of Fungal Colonization on the Provox™ Tracheoesophageal Voice Prosthesis in Post Laryngectomy Patients.

BACKGROUND: Tracheoesophageal voice prostheses (TVPs) have been the gold standard in rehabilitation, after laryngectomy, producing faster and premier voicing towards esophageal speech. Fungal colonization shortens the device's lifetime and leads to prosthesis dysfunction, leakage, and subsequent respiratory infection. Therefore, in the current study, we aimed to investigate the fungal colonization patterns and to propose prophylactic measures that shall increase the longevity of voice prosthesis. METHODS: Failed TVPs were removed - due to leakage and/or aspiration - from 66 post laryngectomy patients and examined. They were referred to Amiralam and Rasoul Hospital, the main centers of Ear, Nose, and Throat in Tehran, Iran from April 2018 to January 2020. Fungal colonization patterns were assessed using DNA sequencing techniques. Furthermore, the susceptibility to fluconazole, amphotericin B, nystatin, and white vinegar was evaluated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: Resident fungal species from the upper airways colonized all the 66 TVPs (100%). Diabetes (31%) and smoking (98%) were the predominant underlying disease and predisposing factors, respectively. Among the 79 fungal agents isolated from the 66 TVPs, Candida glabrata (n=25, 31.7%) was the most common. A significant reduction in minimum inhibitory concentration (MIC) values were observed for white vinegar when used alone (P<0.05). CONCLUSION: White vinegar at a very low concentration could decrease the amount of fungal colonization on TVPs without any adverse effects; its wide accessibility and affordability ensure a decrease in the overall health cost.

First molecular report of causative agent of otomycosis due to Aspergillus luchuensis.

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 ß-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.

The Human Gut Microbe Bacteroides thetaiotaomicron Suppresses Toxin Release from Clostridium difficile by Inhibiting Autolysis.

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.

Effect of thymoquinone on Fusobacterium nucleatum­associated biofilm and inflammation.

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 FN­associated biofilm and inflammation. FN­containing biofilms were prepared by co­cultivation 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 co­aggregation 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 dose­dependently suppressed TNF­α production from a human monocytic cell line, THP­1 exposed to FN, yet showed no toxicity to THP­1 cells. These results indicated that oral hygiene care using TQ could reduce FN­associated biofilm and inflammation in gingival tissue.

Investigation of Etiologic Agents and Clinical Presentations of Otomycosis at a Tertiary Referral Center in Tehran, Iran.

BACKGROUND: Otomycosis is a superficial infection of the ear caused by a spectrum of various fungal agents and its epidemiology depends on geographical region and climatic condition. The aim of this study was to investigate the causal agents and clinical manifestations of otomycosis at a tertiary referral center in Tehran, Iran. METHODS: From Apr 2016 to Jan 2017 a set of 412 subjects with suspicion of external otitis were included. Clinical examination and specimen collection were performed by an otorhinolaryngologist. Subsequently, direct examination and culture were performed on specimens and isolated molds were identified morphologically. Yeast isolates were identified using CHROMagar Candida medium and PCR-RFLP of ribosomal DNA whenever needed. Data were analyzed using SPSS. RESULTS: Otomycosis was confirmed in 117 cases (28.39%) including 64 (54.7%) males and 53 (45.3%) females. Patients were within the age range of 10-75 yr and the highest prevalence was found in the age group of 46-55 yr (30.77%). Pruritus (89.74%) and auditory manipulation and trauma (83.76%) were the predominant symptom and predisposing factor, respectively. Among 133 isolates from 117 patients, Aspergillus niger (n=50, 37.59%) was the most common etiologic agent and Candida glabrata (n=25, 18.8%) was the predominantly isolated yeast. Furthermore, 16 cases of mixed infection were identified and coinfection due to A. niger and C. glabrata (seven cases) was the predominant pattern. CONCLUSION: Our results revealed the high prevalence of C. glabrata and mixed infections in otomycosis patients. Therefore, mycological examinations should be considered for proper treatment.

Ethanolamine utilization supports Clostridium perfringens growth in infected tissues.

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.

Characterization of a recombinant Bacteroides fragilis sialidase expressed in Escherichia coli.

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.

L­histidine augments the oxidative damage against Gram­negative bacteria by hydrogen peroxide.

Excessive damage to DNA and lipid membranes by reactive oxygen species reduces the viability of bacteria. In the present study, the proliferation of recA­deficient Escherichia coli strains was revealed to be inhibited by 1% L­histidine under aerobic conditions. This inhibition of proliferation was not observed under anaerobic conditions, indicating that L­histidine enhances oxidative DNA damage to E. coli cells. Reverse transcription­quantitative polymerase chain reaction analysis demonstrated that the expression of recA in E. coli MG1655 increased ~7­fold following treatment with 10 mM hydrogen peroxide (H2O2) plus 1% L­histidine, compared with that following exposure to H2O2 alone. L­histidine increased the genomic fragmentation of E. coli MG1655 following exposure to H2O2. In addition, L­histidine 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 L­histidine combination. The combination of 100 mM H2O2 and 1.0% L­histidine significantly reduced the number of viable cells of extended­spectrum­ß­lactamase­producing E. coli and multidrug­resistant Pseudomonas aeruginosa, and this treatment was more effective than 100 mM H2O2 alone, but this effect was not observed in methicillin­resistant Staphylococcus aureus or vancomycin­resistant Enterococcus faecium. The combination of L­histidine and H2O2 may be a useful strategy to selectively increase the microbicidal activity of oxidative agents against Gram­negative bacteria.