Explore the vast range of titles available.

Microbes residing in biofilms confer several fold higher antimicrobial resistances than their planktonic counterparts. Compared to monomicrobial biofilms, polymicrobial biofilms involving multiple bacteria, multiple fungi or both are more dominant in nature. Paradoxically, polymicrobial biofilms are less studied. In this study, ocular isolates of Staphylococcus aureu s, S. epidermidis and Candida albicans , the etiological agents of several ocular infections, were used to demonstrate their potential to form mono- and polymicrobial biofilms both in vitro and on human cadaveric corneas. Quantitative (crystal violet and XTT methods) and qualitative (confocal and scanning electron microscopy) methods demonstrated that they form polymicrobial biofilms. The extent of biofilm formation was dependent on whether bacteria and fungi were incubated simultaneously or added to a preformed biofilm. Additionally, the polymicrobial biofilms exhibited increased resistance to different antimicrobials compared to planktonic cells. When the MBECs of different antibacterial and antifungal agents were monitored it was observed that the MBECs in the polymicrobial biofilms was either identical or decreased compared to the monomicrobial biofilms. The results are relevant in planning treatment strategies for the eye. This study demonstrates that ocular bacteria and fungi form polymicrobial biofilms and exhibit increase in antimicrobial resistance compared to the planktonic cells.

Dysbiosis in the gut microbiome has been implicated in several diseases including auto-immune diseases, inflammatory diseases, cancers and mental disorders. Keratitis is an inflammatory disease of the eye significantly contributing to corneal blindness in the developing world. It would be worthwhile to investigate the possibility of dysbiosis in the gut microbiome being associated with Keratitis. Here, we have analyzed fungal and bacterial populations in stool samples through high-throughput sequencing of the ITS2 region for fungi and V3-V4 region of 16S rRNA gene for bacteria in healthy controls (HC, n = 31) and patients with fungal keratitis (FK, n = 32). Candida albicans (2 OTUs), Aspergillus (1 OTU) and 3 other denovo-OTUs were enriched in FK samples and an unclassified denovo-OTU was enriched in HC samples. However, the overall abundances of these 'discriminatory' OTUs were very low (< 0.001%) and not indicative of significant dysbiosis in the fungal community inhabiting the gut of FK patients. In contrast, the gut bacterial richness and diversity in FK patients was significantly decreased when compared to HC. 52 OTUs were significantly enriched in HC samples whereas only 5 OTUs in FK. The OTUs prominently enriched in HC were identified as Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Lachnospira, Mitsuokella multacida, Bacteroides plebeius, Megasphaera and Lachnospiraceae. In FK samples, 5 OTUs affiliated to Bacteroides fragilis, Dorea, Treponema, Fusobacteriaceae, and Acidimicrobiales were significantly higher in abundance. The functional implications are that Faecalibacterium prausnitzii, an anti-inflammatory bacterium and Megasphaera, Mitsuokella multacida and Lachnospira are butyrate producers, which were enriched in HC patients, whereas Treponema and Bacteroides fragilis, which are pathogenic were abundant in FK patients, playing a potential pro-inflammatory role. Heatmap, PCoA plots and functional profiles further confirm the distinct patterns of gut bacterial composition in FK and HC samples. Our study demonstrates dysbiosis in the gut bacterial microbiomes of FK patients compared to HC. Further, based on inferred functions, it appears that dysbiosis in the gut of FK subjects is strongly associated with the disease phenotype with decrease in abundance of beneficial bacteria and increase in abundance of pro-inflammatory and pathogenic bacteria.

The biofilm-forming potential of Staphylococcus aureus and Staphylococcus epidermidis, isolated from patients with Endophthalmitis, was monitored using glass cover slips and cadaveric corneas as substrata. Both the ocular fluid isolates exhibited biofilm-forming potential by the Congo red agar, Crystal violet and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino) carbonyl-2H-tetra-zolium hydroxide (XTT) methods. Confocal microscopy demonstrated that the thickness of the biofilm increased from 4–120 h of biofilm formation. Scanning electron microscopic studies indicated that the biofilms grown on cover slips and ex vivo corneas of both the isolates go through an adhesion phase at 4 h followed by multilayer clumping of cells with intercellular connections and copious amounts of extracellular polymeric substance. Clumps subsequently formed columns and eventually single cells were visible indicative of dispersal phase. Biofilm formation was more rapid when the cornea was used as a substratum. In the biofilms grown on corneas, clumping of cells, formation of 3D structures and final appearance of single cells indicative of dispersal phase occurred by 48 h compared to 96–120 h when biofilms were grown on cover slips. In the biofilm phase, both were several-fold more resistant to antibiotics compared to planktonic cells. This is the first study on biofilm forming potential of ocular fluid S. aureus and S. epidermidis on cadaveric cornea, from attachment to dispersal phase of biofilm formation.

Biofilms confer several advantages to the organisms associated with them, such as increased resistances to antibacterial and antifungal compounds compared to free living cells. Compared to monomicrobial biofilms involving a single microorganism, biofilms composed of microorganisms affiliated to bacterial and fungal kingdoms are predominant in nature. Despite the predominance of polymicrobial biofilms, and more so mixed polymicrobial biofilms, they are rarely studied. The objective of the current study is to evaluate the potential of ocular bacteria and a filamentous fungus to form monomicrobial and mixed polymicrobial biofilms on synthetic and natural substrates and to monitor their response to antibiotics. In this sense, we demonstrated that the ocular pathogens Staphylococcus aureus, S. epidermidis, and Fusarium solani form monomicrobial and mixed polymicrobial biofilms both on tissue culture polystyrene plates and on ex vivo human corneas from cadavers using confocal microscopy and scanning electron microscopy. Additionally, the mixed polymicrobial biofilms involving the above ocular bacteria and a filamentous fungus were less susceptible to different antibacterials and antifungals in relation to the corresponding control planktonic cells. Further, the MICs to the screened antibacterials and antifungals in polymicrobial biofilms involving a bacterium or a fungus was either increased, decreased, or unchanged compared to the corresponding individual bacterial or fungal biofilm. The results would be useful to the ophthalmologist to plan effective treatment regimens for the eye since these are common pathogens of the eye causing keratitis, endophthalmitis, conjunctivitis, etc.

A 55-year-old lady developed a corneal ring infiltrate following trauma with a wooden stick. 10% KOH mount of corneal scrapings revealed septate hyaline fungal filaments. White feathery colonies with shiny black dots grew on potato dextrose agar. Characteristic features of Pestalotiopsis spores were seen on Lactophenol cotton blue mount. DNA sequencing showed 99% similarity with Pseudopestalotiopsis theae. Complete resolution was noted with topical and oral antifungals. To the best of our knowledge, this is the first report of Pseudopestalotiopsis keratitis following trauma with vegetative matter highlighting the role of DNA sequencing in identification of rare fungi.

Purpose: To determine the presence of herpes simplex virus and varicella zoster virus (HSV 1 and 2, VZV) in the cornea of normal subjects by multiplex real time quantitative (qPCR) assay and evaluate its utility in the diagnosis of viral keratitis. Methods: Corneal epithelial cells from 33 eyes of 22 patients undergoing photorefractive keratectomy surgery (controls) and 50 corneal scrapings from 50 patients with suspected HSV keratitis were analyzed for the presence of HSV1 by conventional PCR and for presence of HSV1 and 2 and/or VZV by multiplex real-time PCR. Corneal scrapings of patients were also tested for HSV1 antigen by immunofluorescence assay (IFA). The results were compared and clinical records reviewed. Results: HSV1 and VZV DNA were detected in 8/33 controls (mean-14.3 ± 7.96, range: 3-29.1 copies/mL) and 2/33 controls (mean-10.7 ± 10.9, range 3-18.5 copies/ml) respectively. HSV2 was not detected in any of the controls. Copy numbers above the mean + 1SD of controls were considered significant for viral load in patient samples. Significantly higher number of corneal scrapings (39/50, 78%) from patients were positive for HSV1 (1.2 × 106 copies/mL ± 3.7 × 106 copies/mL) by real time qPCR compared to IFA (11/48, 23%, P value 0.0001) and conventional PCR (20/50, 40%, P value 0.0002). Double infection with HSV-1 (1.5 × 107 copies/ml) and HSV-2 (3.57 × 104 copies/ml) in one case and VZV infection (1.03 × 102 copies/ml) in another was also detected by the multiplex real-time PCR. Conclusion: Multiplex real-time PCR reliably detects HSV1 and 2 and VZV DNA and is ideal for the diagnosis of HSV and VZV keratitis in an ocular microbiology laboratory.

[This corrects the article DOI: 10.1371/journal.pone.0199640.].

Background Escherichia coli, the gastrointestinal commensal, is also known to cause ocular infections such as conjunctivitis, keratitis and endophthalmitis. These infections are normally resolved by topical application of an appropriate antibiotic. But, at times these E. coli are resistant to the antibiotic and this could be due to formation of a biofilm. In this study ocular E. coli from patients with conjunctivitis, keratitis or endophthalmitis were screened for their antibiotic susceptibility and biofilm formation potential. In addition DNA-microarray analysis was done to identify genes that are involved in biofilm formation and antibiotic resistance. Results Out of 12 ocular E. coli isolated from patients ten isolates were resistant to one or more of the nine antibiotics tested and majority of the isolates were positive for biofilm formation. In E. coli L-1216/2010, the best biofilm forming isolate, biofilm formation was confirmed by scanning electron microscopy. Confocal laser scanning microscopic studies indicated that the thickness of the biofilm increased up to 72 h of growth. Further, in the biofilm phase, E. coli L-1216/2010 was 100 times more resistant to the eight antibiotics tested compared to planktonic phase. DNA microarray analysis indicated that in biofilm forming E. coli L-1216/2010 genes encoding biofilm formation such as cell adhesion genes, LPS production genes, genes required for biofilm architecture and extracellular matrix remodeling and genes encoding for proteins that are integral to the cell membrane and those that influence antigen presentation are up regulated during biofilm formation. In addition genes that confer antimicrobial resistance such as genes encoding antimicrobial efflux ( mdt M and cyc A), virulence ( ins Q, yjg K), toxin production ( sat, yjg K, chp S, chp B and ygj N), transport of amino-acids and other metabolites ( cbr B, cbr C, his I and mgl B) are also up regulated. These genes could serve as potential targets for developing strategies for hacking biofilms and overcoming antibiotic resistance. Conclusions This is the first study on global gene expression in antibiotic resistant ocular E. coli with a potential to form biofilm. Using native ocular isolates for antibiotic susceptibility testing, for biofilm formation and global gene expression is relevant and more acceptable than using type strains or non clinical strains which do not necessarily mimic the native isolate.

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.

Biofilms confer several advantages to the organisms associated with them, such as increased resistances to antibacterial and antifungal compounds compared to free living cells. Compared to monomicrobial biofilms involving a single microorganism, biofilms composed of microorganisms affiliated to bacterial and fungal kingdoms are predominant in nature. Despite the predominance of polymicrobial biofilms, and more so mixed polymicrobial biofilms, they are rarely studied. The objective of the current study is to evaluate the potential of ocular bacteria and a filamentous fungus to form monomicrobial and mixed polymicrobial biofilms on synthetic and natural substrates and to monitor their response to antibiotics. In this sense, we demonstrated that the ocular pathogens Staphylococcus aureus, S. epidermidis, and Fusarium solani form monomicrobial and mixed polymicrobial biofilms both on tissue culture polystyrene plates and on ex vivo human corneas from cadavers using confocal microscopy and scanning electron microscopy. Additionally, the mixed polymicrobial biofilms involving the above ocular bacteria and a filamentous fungus were less susceptible to different antibacterials and antifungals in relation to the corresponding control planktonic cells. Further, the MICs to the screened antibacterials and antifungals in polymicrobial biofilms involving a bacterium or a fungus was either increased, decreased, or unchanged compared to the corresponding individual bacterial or fungal biofilm. The results would be useful to the ophthalmologist to plan effective treatment regimens for the eye since these are common pathogens of the eye causing keratitis, endophthalmitis, conjunctivitis, etc.