Intraocular Microbiome in Diabetes and Diabetic Retinopathy: A Pilot Study.

INTRODUCTION: The objective of this study was to compare the microbiome in the aqueous humour and gut of people with diabetes mellitus (DM) with and without diabetic retinopathy (DR). METHODS: This was a prospective controlled study. The study included 17 people undergoing intraocular surgery in their naïve eyes. Stool samples were obtained in the perioperative period; aqueous humour samples of sufficient quantity were obtained in 12 people during intraocular surgery. Dietary information was obtained using a previously validated questionnaire. The gut and aqueous humour samples were assessed for microbiome using 16S rRNA gene sequencing coupled with QIIME and R software. RESULTS: Aqueous humour was analysed in 12 people: 4 each healthy controls, people with DM, and people with DR. There were minor differences at the phyla levels, but the aqueous humour microbiomes of healthy controls, DM, and DR formed three distinct clusters on heat map analysis with discriminatory genera. This genera-level clustering was more apparent for the intraocular than the gut microbiome. In people with DM and DR, we identified genera unique to the eye or the gut. There was a consistent reduction in the abundance of anti-inflammatory bacteria in people with DR than DM. CONCLUSIONS: There is a difference in intraocular and gut microbiome regardless of disease or health. Our preliminary findings indicate distinctive features of the intraocular microbiome in people with DR compared with those without it. While this distinctiveness appears more evident in aqueous humour than in the gut, it needs further confirmation with larger studies.

Gut mycobiome dysbiosis in rats showing retinal changes indicative of diabetic retinopathy.

The current study compared the gut mycobiomes of diabetic rats generated by a streptozotocin chemical challenge, diabetic rats with retinal changes and normal control rats over a period of 4 months. Sustained increase in blood sugar levels (>150 mg/dL) confirmed the induction of diabetes. Histology and immunohistochemistry were used to identify changes in the retinal tissues in the diabetic rats indicative of the animals progressing into diabetic retinopathy. Gut mycobiomes generated using faecal DNA, indicated dysbiosis at the genus level in both diabetic (DM) and diabetic rats with retinal changes (DRC) when compared with the control rats. In Tables 3-6 the specific genera that were significantly increased/decreased in DM1 and DM2 and in DRC1 and DRC2 respectively compared to the respective controls CT1-CT4 rats are listed. Further, the mycobiomes of the DM and DRC rats separated into distinct clusters following heat-map analysis of the discriminating genera. In addition, ß-diversity analysis separated the mycobiomes of DM and DRC rats from that of the control rats, but the mycobiomes of diabetic rats and diabetic rats with retinal changes showed an overlap. Based on the inferred functions of the discriminating genera in the mycobiomes, we speculated that increase in pathogenic fungi might contribute to the inflammatory status both in diabetic rats and rats showing retinal changes.

Elucidating the clinical, microbiological and molecular diagnostic aspects of Macrophomina phaseolina keratitis.

This study reports the clinico-microbiological features of Macrophomina phaseolina keratitis. Clinically diagnosed as microbial keratitis, six patients underwent microbiological evaluation. Fungal culture isolates from cornea were subjected to DNA sequencing of the ITS region, phylogenetic analysis and reconfirmation by polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) of six antifungal drugs were determined by microbroth dilution method against the six isolates. All patients were treated with antifungals. Failed medical therapy necessitated therapeutic penetrating keratoplasty (TPK). Corneal buttons were processed for histopathology. In all patients, the corneal scraping showed septate hyaline fungal filaments. The BLAST analysis for ITS sequences of all six fungal isolates suggested M. phaseolina, however, when limited to sequences from type material, they matched M. pseudophaseolina. Phylogenetic analysis could not differentiate between these two species and clustered in a single clade. PCR assay of specific gene sequence [MpCal (calmodulin)] reconfirmed all isolates as M. phaseolina. The MICs of voriconazole and posaconazole were lowest (0.03 to 2 and 0.1 to 2µg/mL respectively) and all isolates were susceptible to natamycin. Except for case 1, which healed with a scar on treatment, all other cases worsened, despite medical treatment, necessitating TPK. Histopathology of 3 out of 4 buttons showed the presence of fungal filaments. While direct microscopic examination of corneal scrapings is helpful in diagnosis, identification of M. phaseolina in culture is challenging. Although MICs of commonly used antifungals are low response to medical therapy is not encouraging; patients may require TPK for resolution of infection in M. phaseolina keratitis.

DNA sequencing, phylogenetic analysis and specific PCR confirmed Macrophomina phaseolina keratitis in six patients. Although antifungal susceptibility showed the organisms to be susceptible to natamycin five patients did not respond to treatment and needed keratoplasty.

Dysbiosis in the Gut Microbiome in Streptozotocin-Induced Diabetes Rats and Follow-Up During Retinal Changes.

Purpose: To analyze the gut bacterial microbiome of streptozotocin-induced diabetic rats and rats with retinal changes. Methods: Induction of diabetes was confirmed by an increase in blood sugar (>150 mg/dL), and the progression of diabetes with retinal changes was assessed by histology and immunohistochemistry of retinal sections. Microbiomes were generated using fecal DNA, and the V3-V4 amplicons were sequenced and analyzed by QIIME and R. Results: Dysbiosis in the gut microbiome of diabetic rats and diabetic rats with retinal changes was observed at the phylum and genus levels compared with the control rats. Heat-map analysis based on the differentially abundant genera indicated that the microbiomes of controls and diabetic rats separated into two distinct clusters. The majority of the microbiomes in diabetic rats with retinal changes also formed a distinct cluster from the control rats. ß-diversity analysis separated the microbiome of control rats from the microbiome of diabetic rats and diabetic rats with retinal changes, but the microbiomes of diabetic rats and diabetic rats with retinal changes showed an overlap. Functional analysis indicated that the enhanced inflammation in diabetic rats showing retinal changes could be ascribed to a decrease in anti-inflammatory bacteria and an increase in pathogenic and proinflammatory bacteria. Conclusions: This study showed that the gut bacterial microbiome in diabetic rats with retinal changes was different compared with control rats. The results could help develop novel therapeutics for diabetics and diabetic individuals with retinal changes.

Flavobacterium inviolabile sp. nov. isolated from stream water.

A Gram-stain-negative, rod-shaped, aerobic and non-motile bacterium, designated P2-65T, was isolated from Moonsan stream water in the Republic of Korea. The temperature, NaCl concentration and pH ranges for growth of strain P2-65T were 10-37 °C, 0.0-3.0% (w/v) and 6.5-8.5 with optimum growth at 25-30 °C, 0.0-1.0% and 7.0-7.5, respectively. Comparison of 16S rRNA gene sequence showed that strain P2-65T was closely related to Flavobacterium cauense (95.4%) and Flavobacterium cheniae (95.3%). The major fatty acids were iso-C15:0, iso C17:0 3-OH, summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 9 (iso-C17:1 ω9c and/or 10-methyl C16:0) and iso-C15:0 3-OH. The predominant respiratory quinone was menaquinone-6 (MK-6). The major polar lipids detected in the strain were phosphatidylethanolamine, one aminophospholipid, one unidentified aminolipid and one unidentified polar lipid. The G + C content of the genomic DNA was 39.7%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values for strain P2-65T with closely related Flavobacterium species were below 74.8% and 20%, respectively. Based on polyphasic features, strain P2-65T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium inviolabile sp. nov. is proposed. The type strain is P2-65T (= KCTC 62055T = NBRC 112953T).

Phylogenetic Grouping of Human Ocular Escherichia coli Based on Whole-Genome Sequence Analysis.

Escherichia coli is a predominant bacterium in the intestinal tracts of animals. Phylogenetically, strains have been classified into seven phylogroups, A, B1, B2, C, D, E, and F. Pathogenic strains have been categorized into several pathotypes such as Enteropathogenic (EPEC), Enterotoxigenic (ETEC), Enteroinvasive (EIEC), Enteroaggregative (EAEC), Diffusely adherent (DAEC), Uropathogenic (UPEC), Shiga-toxin producing (STEC) or Enterohemorrhagic (EHEC) and Extra-intestinal pathogenic E. coli (ExPEC). E. coli also survives as a commensal on the ocular surface. However, under conditions of trauma and immune-compromised states, E. coli causes conjunctivitis, keratitis, endopthalmitis, dacyrocystitis, etc. The phylogenetic affiliation and the pathotype status of these ocular E. coli strains is not known. For this purpose, the whole-genome sequencing of the 10 ocular E. coli strains was accomplished. Based on whole-genome SNP variation, the ocular E. coli strains were assigned to phylogenetic groups A (two isolates), B2 (seven isolates), and C (one isolate). Furthermore, results indicated that ocular E. coli originated either from feces (enteropathogenic and enterotoxigenic), urine (uropathogenic), or from extra-intestinal sources (extra-intestinal pathogenic). A high concordance was observed between the presence of AMR (Antimicrobial Resistance) genes and antibiotic resistance in the ocular E. coli strains. Furthermore, several virulent genes (fimB to fimI, papB to papX, etc.) and prophages (Enterobacteria phage HK97, Enterobacteria phage P1, Escherichia phage D108 etc.) were unique to ocular E. coli. This is the first report on a whole-genome analysis of ocular E. coli strains.

Alterations in the Ocular Surface Fungal Microbiome in Fungal Keratitis Patients.

Keratitis, an inflammatory disease of the eye, when neglected could lead to sight-threatening complications and ultimately blindness. Globally, over a million people are affected by keratitis annually. Keratitis has a microbial etiology and is caused by bacteria, fungi, viruses, etc. The present study compared the ocular surface fungal microbiome of healthy individuals and individuals with fungal keratitis. Fungal microbiomes from the conjunctival swabs of healthy individuals and from conjunctival swabs and corneal scrapings of individuals with fungal keratitis were generated using ITS2 region amplicons. Microbiomes were sequenced using Illumina MiSeq 2 × 250 base pair chemistry with a paired-end protocol. Based on Alpha diversity indices, phylum and genera level diversity, abundance differences, and heat map analysis, the fungal microbiomes of conjunctival swabs and corneal scrapings of individuals with fungal keratitis exhibited dysbiosis (alterations in the diversity and abundance) compared to the ocular surface microbiome of the healthy control individuals. This is the first report indicating dysbiosis in the fungal microbiome of conjunctival swabs and corneal scrapings in individuals with fungal keratitis. A total of 11 genera present in the majority of the eyes constituted the variable core ocular microbiome.

Sphingobacterium praediipecoris sp. nov. isolated from effluent of a dairy manure treatment plant.

A novel Gram-reaction-negative, rod-shaped, non-motile bacterium, designated as strain G2-10T was isolated from effluent of a dairy manure treatment plant. Growth occurred at 20-40 °C (optimum at 25-30 °C), pH 7.0-8.0 (optimum at pH 7.0). The range of NaCl concentration for growth was between 0% and 3% (w/v) (optimum 0-1%, w/v). Comparison of 16S rRNA gene sequence indicated that strain G2-10T was moderately related to the type strains of Sphingobacterium nematocida M-SX103T and Sphingobacterium suaedae T47T with a pair-wise sequence similarity of 94.3% and 94.0%, respectively. The major fatty acid constituents of strain G2-10T were identified as iso-C15:0 (37.6%), summed feature 3 (consisting of C16:1ω7c and/or C16:1ω6c, 29.6%) and iso-C17:0 3-OH (15.2%). Phosphatidylethanolamine was the major polar lipids of strain G2-10T. Sphingophospholipids were present. The isoprenoid quinone was composed of only MK-7. The DNA G + C content of strain G2-10T was found to be 42.5 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain G2-10T represents a novel species within the genus Sphingobacterium, for which the name Sphingobacterium praediipecoris is proposed. The type strain is G2-10T (= KCTC 52880T = NBRC 112848T).

Flavobacterium amnigenum sp. nov. Isolated from a River.

A yellowish, flexirubin-pigment-producing strain I3-3T was isolated from river water in Iksan, the Republic of Korea. The strain was gram-negative, aerobic, non-motile, showed catalase and oxidase activities, and could grow at a temperature range of 10-35°C, pH 5.0-10 and 0-2.0% (w/v) of NaCl. The major fatty acids were iso-C15:0, iso-C17:0 3-OH and summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c). The isolate contained phosphatidylethanolamine, one aminolipid, and two unidentified lipids as the major polar lipids. Menaquinone-6 (MK6) was the major respiratory quinone. The G+C content of the genomic DNA of strain I3-3T was 35.6%. Comparison of the 16S rRNA gene sequence with the sequences of the closely related type strains showed highest sequence similarity of 96.95% and 96.93% to Flavobacterium nitrogenifigens NXU-44T and Flavobacterium compostarboris 15C3T, respectively. Based on phenotypic and phylogenetic distinctiveness, strain I3-3T is considered as a member of novel species within the genus Flavobacterium, for which Flavobacterium amnigenum sp. nov. is proposed. The type strain is I3-3T (=KCTC 52884T =NBRC 112871T).

Soonwooa purpurea sp. nov., isolated from a fresh water river.

A pale-brown-coloured, rod-shaped, non-motile, and Gram-reaction-negative bacterium, strain I54T, was isolated from a water sample of a freshwater river in Iksan, Republic of Korea. The phylogenetic affiliation based on 16S rRNA gene sequence analysis showed that strain I54T belonged to the genus Soonwooa of the family Flavobacteriaceae with a sequence similarity of 97.5 % to Soonwooa buanensis HM0024T. The major fatty acids (>5 %) of strain I54T were iso-C15 : 0, anteiso-C15 : 0, summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and iso-C17 : 0 3-OH. Phosphatidylethanolamine, one aminolipid and two unknown lipids were the major polar lipids. The G+C content of the genomic DNA was 34.2 (±0.3)mol% and MK6 was the sole respiratory quinone. On the basis of its molecular and phenotypic characteristics, strain I54T represents a novel species in the genus Soonwooa, for which the name Soonwooapurpurea sp. nov. is proposed. The type strain is I54T (=KCTC 52722T=JCM 31880T).

Chitinimonas naiadis sp. nov., Isolated from a Freshwater River.

A rod shaped, aerobic, Gram-stain-negative, and motile bacterium, strain AR2T, was isolated from a water sample of Yeongsan river, Republic of Korea. Strain AR2T clustered closely with the members of the genus Chitinimonas and showed the highest 16S rRNA gene sequence similarity with Chitinimonas prasina LY03T (96.4%), Chitinimonas viridis HMD2169T (96.4%), Chitinimonas taiwanensis cfT (96.2%), and Chitinimonas koreensis R2A43-10T (94.2%). The predominant fatty acids of strain AR2T were identified to be summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c), C16:0, and C10:03-OH. Diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine were found to be the major polar lipids. The genomic DNA G+C content was 60.4 mol%. Based on the polyphasic characterization, the isolated strain AR2T is described as a representative of a novel species in the genus Chitinimonas, for which the name Chitinimonas naiadis sp. nov. (type strain =AR2T =KCTC 42755T =JCM 31504T) is proposed.

Reclassification of Aeromonas sharmana to a new genus as Pseudaeromonas sharmana gen. nov., comb. nov., and description of Pseudaeromonas pectinilytica sp. nov. isolated from a freshwater stream.

A Gram-reaction-negative, rod-shaped, facultatively anaerobic, motile bacterium, designated strain AR1T, was isolated from a freshwater stream in Jeonju, South Korea. Strain AR1T showed highest 16S rRNA gene sequence similarity (96.83 %) and also formed a separate clade with Aeromonas sharmana GPTSA-6T in the phylogenetic tree reconstructed among the members of the family Aeromonadaceae. Major cellular fatty acids are summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and C16: 0. Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol are the predominant polar lipids. The genomic DNA G+C content was found to be 54.7 mol%. However, earlier studies on 16S rRNA gene, gyrB, rpoD and universal target region of cpn60 sequences of the members of the genus Aeromonas recommended the transfer of Aeromonas sharmana to a new genus. Hence, based on the comparative polyphasic data obtained during the present study and also on the previous recommendations, it is proposed that Aeromonas sharmana be transferred to a novel genus as Pseudaeromonas sharmana gen. nov., comb. nov. with strain GPTSA-6T (=DSM 17445T=MTCC 7090T=CIP 109378T=CCUG 54939T) as the type strain of the type species of the genus. Also, it is proposed that strain AR1T be designated as a representative of a novel species of this new genus, namely Pseudaeromonas pectinilytica sp. nov. The type strain is AR1T (=KCTC 42754T=JCM 31503T).

Paenibacillus gelatinilyticus sp. nov. a psychrotolerant bacterium isolated from a reclaimed soil and amended description of Paenibacillus shenyangensis.

A rod shaped, Gram-stain positive, non-motile, facultative anaerobic and gelatin hydrolysing bacterium, strain PG1(T), was isolated from reclaimed land soil in Kyehwa-do, Republic of Korea. Strain PG1(T) showed highest 16S rRNA gene sequence similarity (97.4 and 96.5%, respectively) to Paenibacillus shenyangensis A9(T) and Paenibacillus hunanensis FeL05(T), and clustered closely with the members of the family Paenibacillaceae. DNA-DNA hybridization studies revealed a genomic relatedness of 47 ± 9% with P. shenyangensis A9(T). The predominant fatty acids of strain PG1(T) were identified to be anteiso-C(15:0) (46.7%) and C(16:0) (22.7%). Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid were found to be the major polar lipids. The genomic DNA G+C content was found to be 47.7 mol%. This polyphasic characterisation of the newly isolated strain PG1(T) justifies its description as representative of a novel species in the genus Paenibacillus, for which the name Paenibacillus gelatinilyticus sp. nov., (type strain = PG1(T) = KCTC 33642(T) = JCM 30624(T)) is proposed.