Disturbances in the ocular surface microbiome by perioperative antimicrobial eye drops.

We aimed to elucidate the effects of antimicrobial eye drops used in the perioperative period of ophthalmic surgery on the ocular surface microbiome by metagenomic analysis. Twenty-eight eyes from 15 patients (mean age 74.1 years) with no history of eye drop use within 3 months before cataract surgery were included in this study. Gatifloxacin eye drops were used in all patients in the perioperative period. The antimicrobial eye drops were started 3 days before surgery. They were discontinued after conjunctival sac specimen collection for 2 weeks after the surgery. Conjunctival sac specimens were collected to investigate the alterations in the ocular surface microbiome by meta-16S analysis targeting the V3-V4 region of the bacterial 16S rRNA gene. Principal coordinate analysis showed that the bacterial composition tended to be different before and 2 and 4 weeks after surgery. Individual observations on six eyes showed that the bacterial composition at 12 weeks after surgery was closer to that before surgery than to that at 4 weeks after surgery in two eyes, while the bacterial composition in the remaining four eyes was different at various time points. Before surgery, Firmicutes, Proteobacteria, and Bacteroidetes were predominant; however, 2 weeks after surgery, the proportion of Proteobacteria increased and that of Firmicutes decreased. A similar trend was noticed 4 weeks after surgery, although antibacterial eye drops had been discontinued 2 weeks after surgery. The Shannon-Weaver coefficient showed a decreasing trend at 2-, 4-, and 12-weeks post operation compared to that before operation. The diversity of the microbiome decreased significantly at 2- and 4-weeks after surgery when compared to that before surgery (p < 0.05). The ocular surface microbiome is easily disrupted by antimicrobial eye drops, and it needs recovery time. In such cases, the ocular surface microbiome is presumed to contain many antimicrobial-resistant bacteria. In some cases, it may not recover, and a new microbiome is formed.

Porphyromonas gingivalis Outer Membrane Vesicles Stimulate Gingival Epithelial Cells to Induce Pro-Inflammatory Cytokines via the MAPK and STING Pathways.

Porphyromonas gingivalis (Pg) is a keystone pathogen associated with chronic periodontitis and produces outer membrane vesicles (OMVs) that contain lipopolysaccharide (LPS), gingipains, and pathogen-derived DNA and RNA. Pg-OMVs are involved in the pathogenesis of periodontitis. Pg-OMV-activated pathways that induce the production of the pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in the human gingival epithelial cell line, OBA-9, were investigated. The role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in levels of Pg-OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. Pg-OMVs induced IL-6 and IL-8 production via the extracellular signal-regulated kinase (Erk) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, and NF-κB signaling pathways in OBA-9 cells. In addition, the stimulator of interferon genes (STING), an essential innate immune signaling molecule, was triggered by a cytosolic pathogen DNA. Pg-OMV-induced IL-6 and IL-8 mRNA expression and production were significantly suppressed by STING-specific small interfering RNA. Taken together, these results demonstrated that Pg-OMV-activated Erk1/2, JNK, p38 MAPK, STING, and NF-κB signaling pathways resulting in increased IL-6 and IL-8 expression in human gingival epithelial cells. These results suggest that Pg-OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.

Partially hydrolyzed guar gum alleviates hepatic steatosis and alters specific gut microbiota in a murine liver injury model.

PURPOSE: The gut microbiota, via the gut-liver axis, plays an important role in the development of intestinal failure-associated liver disease. Here, we investigated whether partially hydrolyzed guar gum (PHGG), a dietary fiber could alleviate liver damage and modulate the gut microbiota in a murine liver injury (LI) model. METHODS: Liver injury was induced in 6-week-old male C57BL/6 mice using an enteral liquid diet composed of parenteral nutrition (LI group) and treated with 5% PHGG (LI/PHGG group). Liver histopathology was examined using oil red O and a tumor necrosis factor-α (TNF-α) labeling. The gut microbiota was examined using 16S rRNA gene sequencing. RESULTS: Lipid accumulation was significantly decreased in the LI /PHGG group when compared with that of the LI group. The area of TNF-α-positive cells was significantly higher in the LI group when compared with that of the control. The principal coordinate analysis (PCoA) revealed pronounced changes in the gut microbiota after PHGG treatment. Linear discriminant analysis of effect size showed that PHGG treatment significantly increased cecal abundance of Parabacteroides. CONCLUSIONS: PHGG alleviated hepatic steatosis following liver injury in mice. The protective effect of PHGG treatment could be associated with increased abundance of Parabacteroides in the cecum.

Inactivation of human norovirus by chlorous acid water, a novel chlorine-based disinfectant.

INTRODUCTION: Human norovirus (HuNoV) is a leading cause of infectious gastroenteritis. Since HuNoV shows resistance to alcohol, chlorine-based sanitizers are applied to decontaminate the virus on environmental surfaces. Chlorous acid water (CA) has been recently approved as a novel chlorine-based disinfectant categorized as a Type 2 OTC medicine in Japan. In this study, we aimed to evaluate the capability of CA to inactivate HuNoV. METHODS: HuNoV (genogroups GII.2 and GII.4) was exposed to the test disinfectants including CA and sodium hypochlorite (NaClO), and the residual RNA copy was measured by reverse transcription quantitative PCR (RT-qPCR) after pretreatment with RNase. In addition, the log10 reduction of HuNoV RNA copy number by CA and NaClO was compared in the presence of bovine serum albumin (BSA), sheep red blood cells (SRBC), polypeptone, meat extract or amino acids to evaluate the stability of these disinfectants under organic-matter-rich conditions. RESULTS: In the absence of organic substances, CA with 200 ppm free available chlorine provided >3.0 log10 reduction in the HuNoV RNA copy number within 5 min. Even under high organic matter load (0.3% each of BSA and SRBC or 0.5% polypeptone), 200 ppm CA achieved >3.0 log10 reduction in HuNoV RNA copy number while less than 1.0 log10 reduction was observed with 1,000 ppm sodium hypochlorite (NaClO) in the presence of 0.5% polypeptone. CA reacted with only cysteine, histidine and glutathione while NaClO reacted with all of the amino acids tested. CONCLUSIONS: CA is an effective disinfectant to inactivate HuNoV under organic-matter-rich conditions.

MetaPlatanus: a metagenome assembler that combines long-range sequence links and species-specific features.

De novo metagenome assembly is effective in assembling multiple draft genomes, including those of uncultured organisms. However, heterogeneity in the metagenome hinders assembly and introduces interspecies misassembly deleterious for downstream analysis. For this purpose, we developed a hybrid metagenome assembler, MetaPlatanus. First, as a characteristic function, it assembles the basic contigs from accurate short reads and then iteratively utilizes long-range sequence links, species-specific sequence compositions, and coverage depth. The binning information was also used to improve contiguity. Benchmarking using mock datasets consisting of known bacteria with long reads or mate pairs revealed the high contiguity MetaPlatanus with a few interspecies misassemblies. For published human gut data with nanopore reads from potable sequencers, MetaPlatanus assembled many biologically important elements, such as coding genes, gene clusters, viral sequences, and over-half bacterial genomes. In the benchmark with published human saliva data with high-throughput nanopore reads, the superiority of MetaPlatanus was considerably more evident. We found that some high-abundance bacterial genomes were assembled only by MetaPlatanus as near-complete. Furthermore, MetaPlatanus can circumvent the limitations of highly fragmented assemblies and frequent interspecies misassembles obtained by the other tools. Overall, the study demonstrates that MetaPlatanus could be an effective approach for exploring large-scale structures in metagenomes.

MicroRNA-based host response to toxicant exposure is influenced by the presence of gut microbial populations.

Segmented filamentous bacteria (SFB) and Bacteroides fragilis are known to interact with the host immune response through the aryl hydrocarbon receptor (Ahr). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental toxicant and a high-affinity Ahr ligand has the potential to modify the effect of SFB and B. fragilis. MicroRNAs (miRNA) with their role in regulating gene expression post-transcriptionally, may potentially be used to observe such interactions between SFB, B. fragilis, and TCDD. However, little is known regarding the impact of gut microbial members on miRNA expression or its modulation in the presence of an environmental toxicant. This information is important in understanding toxicant-mediated dysbiosis in gut microbiome and the resulting human health impacts. In this study, C57BL/6 germ-free (GF) mice were colonized with SFB and B. fragilis and administered 30 µg/kg TCDD every 4 d for 28 d and miRNA were measured. Compared to GF mice, colonization with SFB resulted in an increase in up- and down-regulated Ileal miRNAs. TCDD treatment of this group decreased the number of upregulated miRNA and increased the number of down-regulated miRNAs. Association with SFB and B. fragilis together had a similar but less pronounced effect in response to TCDD treatment. TCDD treatment of GF mice had no miRNA expression response. Immune and inflammatory responses and T-cell differentiation were the key functions impacted by these miRNAs. Overall, these results reveal that the host response to toxicants may also depend on the presence of specific gut microbial populations.

Parabacteroides distasonis: intriguing aerotolerant gut anaerobe with emerging antimicrobial resistance and pathogenic and probiotic roles in human health.

Parabacteroides distasonis is the type strain for the genus Parabacteroides, a group of gram-negative anaerobic bacteria that commonly colonize the gastrointestinal tract of numerous species. First isolated in the 1930s from a clinical specimen as Bacteroides distasonis, the strain was re-classified to form the new genus Parabacteroides in 2006. Currently, the genus consists of 15 species, 10 of which are listed as 'validly named' (P. acidifaciens, P. chartae, P. chinchillae, P. chongii, P. distasonis, P. faecis, P. goldsteinii, P. gordonii, P. johnsonii, and P. merdae) and 5 'not validly named' (P. bouchesdurhonensis, P. massiliensis, P. pacaensis, P. provencensis, and P. timonensis) by the List of Prokaryotic names with Standing in Nomenclature. The Parabacteroides genus has been associated with reports of both beneficial and pathogenic effects in human health. Herein, we review the literature on the history, ecology, diseases, antimicrobial resistance, and genetics of this bacterium, illustrating the effects of P. distasonis on human and animal health.

Identifying the chloroperoxyl radical in acidified sodium chlorite solution.

The present study identified the active radical species in acidic sodium chlorite and investigated the feasibility of quantifying these species with the diethylphenylenediamine (DPD) method. Electron spin resonance (ESR) spectroscopy was used to identify the active species generated in solutions containing sodium chlorite (NaClO2). The ESR signal was directly observed in an acidified sodium chlorite (ASC) aqueous solution at room temperature. This ESR signal was very long-lived, indicating that the radical was thermodynamically stable. The ESR parameters of this signal did not coincide with previously reported values of the chlorine radical (Cl●) or chlorine dioxide radical (O = Cl●-O and O = Cl-O●). We refer to this signal as being from the chloroperoxyl radical (Cl-O-O●). Quantum chemical calculations revealed that the optimal structure of the chloroperoxyl radical is much more thermodynamically stable than that of the chlorine dioxide radical. The UV-visible spectrum of the chloroperoxyl radical showed maximum absorbance at 354 nm. This absorbance had a linear relationship with the chloroperoxyl radical ESR signal intensity. Quantifying the free chlorine concentration by the DPD method also revealed a linear relationship with the maximum absorbance at 354 nm, which in turn showed a linear relationship with the chloroperoxyl radical ESR signal intensity. These linear relationships suggest that the DPD method can quantify chloroperoxyl radicals, which this study considers to be the active species in ASC aqueous solution.

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.

Microbiome analysis of contact lens care solutions and tear fluids of contact lens wearers: Possible involvement of streptococcal antigens in allergic symptoms related to contact lens wear.

The present study elucidated the pathogenesis of allergic symptoms (AS) related to contact lens (CL) wear by assaying CL care solutions in lens storage cases and tears from subjects with AS using molecular biology techniques. A total of 15 CL storage cases were collected from subjects with AS (n=9) and healthy, asymptomatic control CL wearers (n=6). Bacterial populations in CL care solutions and tears were assayed by culture and 16S rDNA sequencing. Histamine levels in tears were measured by high­performance liquid chromatography. Western blot analysis was performed to identify the bacteria recognized by tear IgE from subjects with AS. No significant differences were found in the culture results between the subjects with AS and asymptomatic subjects. Histamine was detected in 2 subjects with AS. Meta­16S rDNA sequencing identified a cluster of 4 subjects with AS that were distinguished from others by principal coordinate analysis. Detailed population analysis revealed that the abundance of Gram­positive bacteria in the microbiomes of CL care solutions used by the subjects with AS were higher than those of asymptomatic subjects (42.24±9.47 vs. 16.85±22.76% abundance). Among these, Streptococcus was the dominant genus (12.1­18.3% abundance). Tear microbiome analysis revealed that the abundance of Streptococcus in the subjects with AS was significantly higher than that in other subjects (19.02±5.50 vs. 3.08±3.35%, P<0.01). Western blot analysis demonstrated that the tear IgE in all subjects with AS reacted with Streptococcus (100%), but not with Staphylococcus. On the whole, these results provide novel insight into the pathogenesis of AS and identify Streptococcus as an important factor in AS associated with CL wear.

D-Tagatose Effectively Reduces the Number of Streptococcus mutans and Oral Bacteria in Healthy Adult Subjects: A Chewing Gum Pilot Study and Randomized Clinical Trial.

We examined the effect of D-Tagatose on the growth of oral bacteria including Streptococcus mutans (S. mutans). Saliva collected from 10 healthy volunteers was plated on BHI medium (to culture total oral bacteria) and MBS medium (to culture S. mutans, specifically). Agar plates of BHI or MBS containing xylitol or D-Tagatose were cultured under aerobic or anaerobic conditions. We then counted the number of colonies. In BHI plates containing D-Tagatose, a complete and significant reduction of bacteria occurred under both aerobic and anaerobic conditions. In MSB medium, significant reduction of S. mutans was also observed. We then performed a doubleblind parallel randomized trial with 19 healthy volunteers. They chewed gum containing xylitol, D-Tagatose, or both for 4 weeks, and their saliva was collected weekly and plated on BHI and MSB media. These plates were cultured under anaerobic conditions. Total bacteria and S. mutans were not effectively reduced in either the D-Tagatose or xylitol gum group. However, S. mutans was significantly reduced in volunteers chewing gum containing both D-Tagatose and xylitol. Thus, D-Tagatose inhibited the growth of S. mutans and many types of oral bacteria, indicating that D-Tagatose intake may help prevent dental caries, periodontitis, and many oral diseases.

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.

Presence of Acanthamoeba and diversified bacterial flora in poorly maintained contact lens cases.

Acanthamoeba can cause visually destructive Acanthamoeba keratitis (AK) in contact lens (CL) users. The purpose of this study was to determine whether Acanthamoeba was present in the CL cases of CL wearers and to develop techniques to prevent the contaminations. To accomplish this, 512 CL case samples were collected from 305 healthy CL wearers. Using real-time PCR, Acanthamoeba DNA was detected in 19.1% of CL cases, however their presence was not directly associated with poor CL case care. Instead, the presence of Acanthamoeba DNA was associated with significant levels of many different bacterial species. When the CL cases underwent metagenomic analysis, the most abundant bacterial orders were Enterobacteriales followed by Burkholderiales, Pseudomonadales, and Flavobacteriales. The presence of Acanthamoeba was characterized by Propionibacterium acnes and Rothia aeria and was also associated with an increase in the α diversity. Collectively, Acanthamoeba contamination occurs when a diversified bacterial flora is present in CL cases. This can effectively be prevented by careful and thorough CL case care.

Antiviral and Virucidal Activities of Umesu Phenolics on Influenza Viruses.

In this study, umesu phenolics were purified from the salt extracts of Japanese apricot (Nanko-mume cultivar of Prunus mume Sieb. et Zucc.). Characterization of umesu phenolics revealed that, when added to the culture media of the infected cells, they inhibited the multiplication of influenza and many other RNA and DNA viruses. In addition to these antiviral activities, the phenolics significantly decreased the plating efficiency of influenza virus, if present in the virus inoculum. More drastic effects were observed in terms of virucidal activity; the infectivity of several strains of influenza viruses decreased less than 0.001 when they were incubated with 4 mg/ml phenolics at 30 ℃ for 5 min. The virucidal activity of phenolics was found to be more remarkable in acidic conditions; however, the activity was not merely a result of the acidity of the phenolics. These results clearly support the antiviral and virucidal activities of the umesu phenolics against influenza viruses and suggest their potential pharmacological usefulness as disinfectants or preventive medicine against superficial infections, such as the respiratory infections.

Partially hydrolyzed guar gum alleviates small intestinal mucosal damage after massive small bowel resection along with changes in the intestinal microbiota.

PURPOSE: Short bowel syndrome is associated with intestinal mucosal inflammation and microbial dysbiosis, leading to intractable complications. Partially hydrolyzed guar gum (PHGG) has trophic and anti-inflammatory effects on the intestine. We investigated whether PHGG ameliorates small intestinal mucosal damage and alters the intestinal microbiota using a rat small bowel resection (SBR) model. METHODS: Sprague Dawley rats were divided into sham operation (Sham), Sham/PHGG, SBR, and SBR/PHGG groups. On day 21, all rats were euthanized. To assess small intestinal mucosal damage, the degeneration rate was morphometrically evaluated and immunohistochemically examined using anti-CD45 antibodies. Analyses of fecal microbiota using 16S rRNA and short-chain fatty acid production were also performed. RESULTS: The mucosal degeneration rate was significantly higher in the SBR group than in the Sham or SBR/PHGG groups. The number of CD45-positive cells was significantly higher in the SBR group than in the Sham, Sham/PHGG, or SBR/PHGG groups. The relative abundance of family Lachnospiraceae was significantly higher in the SBR/PHGG group than in the SBR group. CONCLUSIONS: PHGG administration alleviated small intestinal mucosal damage which could be associated with modulation of the intestinal microbiota.

Ingested d-Aspartate Facilitates the Functional Connectivity and Modifies Dendritic Spine Morphology in Rat Hippocampus.

d-Aspartate (d-Asp), the stereoisomer of l-aspartate, has a role in memory function in rodents. However, the mechanism of the effect of d-Asp has not been fully understood. In this study, we hypothesized that ingested d-Asp directly reaches the hippocampal tissues via the blood circulation and modifies the functional connectivity between hippocampus and other regions through spinogenesis in hippocampal CA1 neurons. The spinogenesis induced by the application of d-Asp was investigated using rat acute hippocampal slices. The density of CA1 spines was increased following 21 and 100 µM d-Asp application. The nongenomic spine increase pathway involved LIM kinase. In parallel to the acute slice study, brain activation was investigated in awake rats using functional MRI following the intragastric administration of 5 mM d-Asp. Furthermore, the concentration of d-Asp in the blood serum and hippocampus was significantly increased 15 min after intragastric administration of d-Asp. A functional connectivity by awake rat fMRI demonstrated increased slow-frequency synchronization in the hippocampus and other regions, including the somatosensory cortex, striatum, and the nucleus accumbens, 10-20 min after the start of d-Asp administration. These results suggest that ingested d-Asp reaches the brain through the blood circulation and modulates hippocampal neural networks through the modulation of spines.

An enhanced green fluorescence protein (EGFP)-based reporter assay for quantitative detection of sporulation in Clostridium perfringens SM101.

Clostridium perfringens type F is a spore-forming anaerobe that causes bacterial food-borne illness in humans. The disease develops when ingested vegetative cells reach the intestinal tract and begin to form spores that produce the diarrheagenic C. perfringens enterotoxin (CPE). Given that CPE production is regulated by the master regulator of sporulation (transcription factor Spo0A), the identification of sporulation-inducing factors in the intestine is relevant to better understanding of the disease. To examine these factors, we established assays to quantify C. perfringens sporulation stage under microscopy by using two fluorescent reporters, namely, Evoglow-Bs2 and CpEGFP. When the reporter genes were placed under control of the cpe promoter, both protein products were expressed specifically during sporulation. However, the intensity of the anaerobic reporter Evoglow-Bs2 was weak and rapidly photobleached during microscopic observation. Alternatively, CpEGFP, a canonical green fluorescence protein with optimized codon usage for Clostridium species, was readily detectable in the mother-cell compartment of most bacteria at early stages of sporulation. Additionally, CpEGFP expression predicted final spore yield and was quantifiable in 96-well plates using fluorescence plate reader. These results indicate that CpEGFP can be used to analyze the sporulation of C. perfringens and has a potential application in the large-scale screening of sporulation-regulating biomolecules.

Genetic diversity and persistent colonization of Enterococcus faecalis on ocular surfaces.

PURPOSE: Enterococcus faecalis causes severe acute endophthalmitis and often leads to poor visual outcomes. Conjunctival bacterial cultures occasionally grow atypical bacteria including E. faecalis, which can potentially contribute to the development of postoperative endophthalmitis. However, the characteristics of these ocular E. faecalis strains are unknown. This study is the first attempt to determine the population characteristics of E. faecalis clinical isolates from eye infections and ocular commensals. STUDY DESIGN: Retrospective METHODS: Twenty-eight E. faecalis ocular isolates were collected from 23 patients at 3 referring hospitals. The multilocus sequence typing (MLST) data were analyzed using the eBURST program. Phenotypes of cytolysin and gelatinase, antibiotic susceptibility, and mutations of the quinolone resistance-determining regions (QRDRs) of gyrA and parC were also examined. Pulsed-field gel electrophoresis (PFGE) was performed for strains from the same patients. RESULTS: PFGE revealed that 3 patients retained identical strains for 10 months to 2 and a half years. MLST identified 12 sequence types (STs), which were clustered into 3 clonal complexes (CCs) and 8 singletons, with ST179 the largest. Thirteen of the 23 isolates (56.5%) belonged to CC58, CC8, or CC2, which have previously been reported to be major CCs. Six of the 23 strains (26.0%) exhibited high-level quinolone resistance derived from mutations of the QRDRs in both gyrA and parC. CONCLUSIONS: The sequence types of E. faecalis ocular isolates were divergent, with no eye-specific lineages observed. Persistent colonization of E. faecalis on the ocular surface was demonstrated in patients with chronic ocular surface diseases.

IgA regulates the composition and metabolic function of gut microbiota by promoting symbiosis between bacteria.

Immunoglobulin A (IgA) promotes health by regulating the composition and function of gut microbiota, but the molecular requirements for such homeostatic IgA function remain unknown. We found that a heavily glycosylated monoclonal IgA recognizing ovalbumin coats Bacteroides thetaiotaomicron (B. theta), a prominent gut symbiont of the phylum Bacteroidetes. In vivo, IgA alters the expression of polysaccharide utilization loci (PUL), including a functionally uncharacterized molecular family provisionally named Mucus-Associated Functional Factor (MAFF). In both mice and humans, MAFF is detected predominantly in mucus-resident bacteria, and its expression requires the presence of complex microbiota. Expression of the MAFF system facilitates symbiosis with other members of the phylum Firmicutes and promotes protection from a chemically induced model of colitis. Our data reveal a novel mechanism by which IgA promotes symbiosis and colonic homeostasis.

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.