Impact of Sample Collection Order on the Diagnostic Performance of Metagenomic Deep Sequencing for Infectious Keratitis.

PURPOSE: The purpose of this article was to evaluate the impact of sample collection order on the diagnostic yield of metagenomic deep sequencing (MDS) for determining the causative pathogen in infectious keratitis. METHODS: We performed a cross-sectional diagnostic test evaluation among subjects with infectious keratitis at Aravind Eye Hospital in Madurai, India. All subjects underwent corneal scrapings of the affected eye to obtain potassium hydroxide smear, Gram stain, bacterial culture, and fungal culture, in this order. The order of MDS specimen collection relative to smear and culture samples was randomized and served as the primary predictor. Outcomes included the normalized copy number of pathogenic RNA detected by MDS, the proportion of MDS samples that were diagnostic, and the agreement of MDS results with cultures. RESULTS: MDS samples from 46 subjects with corneal ulcers were evaluated. MDS was positive in 33 subjects (76%) and had 74% overall agreement with culture results. There was no association between order of MDS sample collection and normalized copy number of genetic material detected (P = 0.62) or the likelihood of MDS positivity (P = 0.46). However, the likelihood of agreement between MDS and cultures decreased when MDS corneal swabs were collected after other diagnostic corneal scrapings (P = 0.05). CONCLUSIONS: The overall yield of MDS for detecting the cause of infectious keratitis was not affected by sample collection order. However, diagnostic agreement between MDS and cultures decreased when MDS samples were collected after other specimens. Additional investigation is warranted to determine whether this represents increased sensitivity of MDS compared with cultures or higher susceptibility to contaminants.

Prophylactic Knockdown of the miR-183/96/182 Cluster Ameliorates Pseudomonas aeruginosa-Induced Keratitis.

Purpose: Previously, we demonstrated that miR-183/96/182 cluster (miR-183C) knockout mice exhibit decreased severity of Pseudomonas aeruginosa (PA)-induced keratitis. This study tests the hypothesis that prophylactic knockdown of miR-183C ameliorates PA keratitis indicative of a therapeutic potential. Methods: Eight-week-old miR-183C wild-type and C57BL/6J inbred mice were used. Locked nucleic acid-modified anti-miR-183C or negative control oligoribonucleotides with scrambled sequences (NC ORNs) were injected subconjunctivally 1 day before and then topically applied once daily for 5 days post-infection (dpi) (strain 19660). Corneal disease was graded at 1, 3, and 5 dpi. Corneas were harvested for RT-PCR, ELISA, immunofluorescence (IF), myeloperoxidase and plate count assays, and flow cytometry. Corneal nerve density was evaluated in flatmounted corneas by IF staining with anti-ß-III tubulin antibody. Results: Anti-miR-183C downregulated miR-183C in the cornea. It resulted in an increase in IL-1ß at 1 dpi, which was decreased at 5 dpi; fewer polymorphonuclear leukocytes (PMNs) at 5 dpi; lower viable bacterial plate count at both 1 and 5 dpi; increased percentages of MHCII+ macrophages (Mϕ) and dendritic cells (DCs), consistent with enhanced activation/maturation; and decreased severity of PA keratitis. Anti-miR-183C treatment in the cornea of naïve mice resulted in a transient reduction of corneal nerve density, which was fully recovered one week after the last anti-miR application. miR-183C targets repulsive axon-guidance receptor molecule Neuropilin 1, which may mediate the effect of anti-miR-183C on corneal nerve regression. Conclusions: Prophylactic miR-183C knockdown is protective against PA keratitis through its regulation of innate immunity, corneal innervation, and neuroimmune interactions.

Characterization of antibiotic resistance and virulence genes of ocular methicillin-resistant Staphylococcus aureus strains through complete genome analysis.

Virulence-factor encoding genes (VFGs) and antimicrobial resistance genes (ARGs) of ocular Methicillin-Resistant Staphylococcus aureus (MRSA), are the reason behind the common cause of severe and untreatable ocular infection and are largely unknown. The unavailability of the complete genome sequence of ocular MRSA strains hinders the unambiguous determination of ARGs and VRGs role in disease pathogenesis and their genomic location. To fulfill this critical need, we achieved the high-quality complete genome of four ocular MRSA strains (AMRF3 - AMRF6) by combining MinION nanopore sequencing technology, followed by polishing with Illumina sequence reads. We obtained a single chromosome and a plasmid in each strain. Sequence typing revealed that AMRF3 and AMRF5 strains harbored ST772, whereas AMRF4 and AMRF6 harbored ST 2066. All plasmids carried heavy metal cadmium resistance genes cadC and cadD, while cadA was detected only in the plasmid pSaa6159 of AMRF4 and AMRF6 strains. Further, pSaa6159 contains a complete Tn552 transposon with beta-lactamase genes, blaI, blaR1, and blaZ. Interestingly, pSaa6159 in AMRF6 carried five copies of Tn552 transposon. Several exotoxins and enterotoxins were identified across ocular MRSA strains and ST2066 strains found to be not carried any enterotoxins; this finding suggests that these two strains are exotoxigenic. Besides, ST2066 strains carried serine proteases (splA, splB, splD, splE and spIF) and exotoxin (seb and set 21) for their virulence, while ST772 carried antimicrobial resistance genes (blaZ, dfrG, msrA, mphC and fosB) and enterotoxin sec for virulence, suggesting sequence type-specific resistance and virulence. Also, we identified many VFGs and ARGs, that provided multi-drug resistance, enterotoxigenic, exotoxigenic, biofilm-forming, host tissue adhesion and immune response evasion in ocular MRSA strains. Thus, our study provides a better insight into the genomes of ocular MRSA strains that would provide more effective treatment strategies for ocular MRSA infection.

Understanding the Interactions Between the Ocular Surface Microbiome and the Tear Proteome.

Purpose: The purpose of this study was to explore the interplay between the ocular surface microbiome and the tear proteome in humans in order to better understand the pathogenesis of ocular surface-associated diseases. Methods: Twenty eyes from 20 participants were included in the study. The ocular surface microbiome was sequenced by whole-metagenome shotgun sequencing using lid and conjunctival swabs. Furthermore, the tear proteome was identified using chromatography tandem mass spectrometry. After compositional and functional profiling of the metagenome and functional characterization of the proteome by gene ontology, association studies between the ocular microbiome and tear proteome were assessed. Results: Two hundred twenty-nine taxa were identified with Actinobacteria and Proteobacteria being the most abundant phyla with significantly more Propionibacterium acnes and Staphylococcus epidermidis in lid compared to conjunctival swabs. The lid metagenomes were enriched in genes of the glycolysis lll and adenosine nucleotides de novo and L-isoleucine biosynthesis. Correlations between the phylum Firmicutes and fatty acid metabolism, between the genus Agrobacterium as well as vitamin B1 synthesis and antimicrobial activity, and between biosynthesis of heme, L-arginine, as well as L-citrulline and human vision were detected. Conclusions: The ocular surface microbiome was found to be associated with the tear proteome with a role in human immune defense. This study has a potential impact on the development of treatment strategies for ocular surface-associated diseases.

Current knowledge on the human eye microbiome: a systematic review of available amplicon and metagenomic sequencing data.

Insights in the ocular surface microbiome are still at an early stage and many more questions remain unanswered compared with other human-associated microbial communities. The current knowledge on the human microbiome changed our viewpoint on bacteria and human health and significantly enhanced our understanding of human pathophysiology. Also in ocular medicine, microbiome research might impact treatment. Here, we summarize the current knowledge on ocular microbiome research with a particular focus on potential confounding factors and their effects on microbiome composition. Moreover, we present the ocular surface core microbiome based on current available data and defined it as genera present in almost half of the published control cohorts with a relative abundance of at least 1%.

Expression of Cytokines in Porcine Iris, Retina and Choroidal Tissues Stimulated by Microbe-associated Molecular Patterns.

PURPOSE: The innate immune system is strongly implicated in the pathogenesis of uveitis. This study was designed to clarify the responses of the innate immune system in uveal tissues. MATERIALS AND METHODS: We utilized quantitative, real-time RT-PCR to measure mRNA of innate immune system receptors from porcine iris, choroid, and retina tissues. We used RT-PCR for cytokines to evaluate the responses of these tissues to specific ligands or extracts of whole bacteria that activate the innate immune system. We used ELISA for IL-6 on selected choroidal supernatants to confirm that the mRNA measurement correlated with protein levels. RESULTS: In each of the studied tissues, we detected the expression of important receptors belonging to the innate immune system including dectin-1, TLR4, TLR8, and NOD2. Relative mRNA expression was generally lower in the retina compared to iris or choroid. All three tissues demonstrated upregulation of cytokine mRNA in response to a range of ligands that activate the innate immune system. The measurement of IL-6 protein was consistent with results based on mRNA. Notably, the expression of mRNA for IL-23 was more pronounced than IL-12 in all three tissues after stimulation with various innate immune system ligands. CONCLUSIONS: These data provide evidence of a potent innate immune response intrinsic to uveal tissues. Specific innate immune system ligands as well as bacterial extracts enhanced the production of several inflammatory cytokines. Furthermore, the observation of higher upregulation of IL-23 mRNA, compared to IL-12 in response to innate immune stimuli, suggested that a local TH17 response might be more robust than a local TH1 response in uveal tissues. Our results expand the understanding as to how the innate immune system may contribute to uveitis.

Application of Metagenomic Sequencing in the Diagnosis of Infectious Uveitis.

Purpose: to summarize the origin and very recent history of the use of metagenomic sequencing for the diagnosis of infectious uveitis, convey the technique as described by one of the primary institutions experimenting with the technology, and present recent successful applications of the technology as well as potential advantages and pitfalls compared to other current diagnostic tools.Methods: review of peer-reviewed literature concerning metagenomic sequencing for the diagnosis of infectious uveitis.Conclusion: compared to existing diagnostic methods, metagenomic deep sequencing is a sensitive, unbiased, and comprehensive technique with great potential for diagnosing the causative pathogens of cases of infectious uveitis. However, many issues remain to be addressed in the process of developing this technology, including but not limited to the potentially overwhelming amount of information generated, definition of diagnostic thresholds, demonstration of validity, contamination, and cost.

RNA-Seq analysis of differentially expressed genes of Staphylococcus epidermidis isolated from postoperative endophthalmitis and the healthy conjunctiva.

Staphylococcus epidermidis (S. epidermidis) is one of the primary pathogens in postoperative endophthalmitis, which is a devastating complication of cataract surgery and often results in irreversible visual loss and even blindness. Meanwhile, it is the most frequently isolated commensal bacterium in the healthy conjunctiva. In this study, we investigated the differentially expressed genes (DEGs) of S. epidermidis isolated from the patients with postoperative endophthalmitis and the healthy conjunctiva to predict their functions and pathways by Illumina high-throughput RNA sequencing. Using genome-wide transcriptional analysis, 281 genes (142 upregulated and 139 downregulated genes) were found to be differentially expressed (fold change ≥ 2, p ≤ 0.05) in the strains from endophthalmitis. Ten randomly selected DEGs were further validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). GO enrichment analysis suggested that more DEGs were associated with the thioredoxin system and iron ion metabolism. KEGG pathway analysis revealed that more DEGs were associated with the pathways of the two-component system and pyruvate metabolism. Moreover, the gene SE1634 code for staphylococcal toxin was significantly upregulated in S. epidermidis strains of the endophthalmitis, which might be directly responsible for the pathogenesis of endophthalmitis. In conclusion, this research is helpful for further investigations on genes or pathways related with the pathogenesis and therapeutic targets of S. epidermidis endophthalmitis.

Prognostic Utility of Whole-Genome Sequencing and Polymerase Chain Reaction Tests of Ocular Fluids in Postprocedural Endophthalmitis.

PURPOSE: To associate detection of potential pathogen DNA in endophthalmitis with clinical outcomes. DESIGN: Prospective cohort study. METHODS: Patients in whom endophthalmitis was diagnosed following an intraocular procedure were recruited. Clinical outcome data from baseline, week-1, month-1, and month-3 visits were collected. Intraocular biopsy samples were cultured by standard methods. Quantitative polymerase chain reaction (qPCR) was performed for specific pathogens and whole-genome sequencing (WGS). RESULTS: A total of 50 patients (mean age 72 years old; 52% male) were enrolled. Twenty-four cases were culture-positive and 26 were culture-negative. WGS identified the cultured organism in 76% of culture-positive cases and identified potential pathogens in 33% of culture-negative cases. Month-1 and -3 visual acuities did not vary by pathogen-positive versus pathogen-negative cases as detected by either culture or WGS. Visual outcomes of Staphylococcus epidermidis endophthalmitis were no different than those of pathogen-negative cases, whereas the patients infected with other pathogens showed worse outcome. Higher baseline bacterial DNA loads of bacteria other than those of S epidermidis detected by WGS were associated with worse month-1 and -3 visual acuity, whereas the S epidermidis loads did not appear to influence outcomes. Torque teno virus (TTV) and Merkel cell polyomavirus (MCV) were detected by qPCR in 49% and 19% of cases, respectively. Presence of TTV at presentation was associated with a higher rate of secondary pars plana vitrectomy (P = .009) and retinal detachment (P = .022). CONCLUSIONS: The presence and higher load of bacteria other than S epidermidis detected by WGS or DNA from TTV by qPCR in ocular fluids is associated with worse outcomes in post-procedure endophthalmitis.

Effectiveness of 16S ribosomal DNA real-time PCR and sequencing for diagnosing bacterial keratitis.

PURPOSE: To evaluate the efficacy of real-time PCR for 16S ribosomal DNA (16S r-DNA) and sequencing for diagnosing microbial keratitis. METHODS: We studied 272 eyes of 272 patients with keratitis. Eyes with keratitis were classified as "definite" (N = 118), "likely" (N = 71), or "non-bacterial" (N = 83) to have bacterial keratitis. The diagnostic efficacy of real-time PCR and conventional testing was determined by receiver operating characteristic analysis. The copy numbers of bacterial DNA and clinical characteristics were retrospectively analyzed for association with concordant culture results in the "definite" cases. RESULTS: The level of bacterial DNA was significantly associated with the diagnostic probability of the three diagnostic categories. The level of bacterial DNA had comparable diagnostic efficacy with the area under the curve (AUC) at 0.67, by culture at 0.65, and by smear testing at 0.73. The efficacy was significantly improved by combining the DNA level with the conventional culture testing with an AUC of 0.81. Analysis of the "definite" cases showed culture positivity in 51.8% (58 eyes), and of these, 41 eyes (70.7%) were higher than the cutoff PCR values and 40 eyes were identified by 16S r-DNA sequencing. In the culture-negative eyes, the level of bacterial DNA was significantly lower (P = 0.0008). Eyes with higher bacterial DNA levels had significantly concordant outcomes with sequencing and culture results (P = 0.006). Previous antibiotic treatments decreased the bacterial DNA amount by 0.09-fold, and it was a significant factor for discordance (P = 0.006). CONCLUSION: Quantification of the bacterial DNA level and conventional testing improves the diagnostic efficacy of infectious bacterial keratitis.

Analysis of differentially expressed genes in bacterial and fungal keratitis.

Purpose: This study was aimed at identifying differentially expressed genes (DEGs) in bacterial and fungal keratitis. The candidate genes can be selected and quantified to distinguish between causative agents of infectious keratitis to improve therapeutic outcomes. Methods: The expression profile of bacterial or fungal infection, and normal corneal tissues were downloaded from the Gene Expression Omnibus. The limma package in R was used to screen DEGs in bacterial and fungal keratitis. The Co-Express tool was used to calculate correlation coefficients of co-expressed genes. The "Advanced network merge" function of Cytoscape tool was applied to obtain a fusional co-expression network based on bacterial and fungal keratitis DEGs. Finally, functional enrichment analysis by DAVID software and KEGG analysis by KOBAS of DEGs in fusion network were performed. Results: In total, 451 DEGs in bacterial keratitis and 353 DEGs in fungal keratitis were screened, among which 148 DEGs were found only in bacterial keratitis and 50 DEGs only in fungal keratitis. Besides, 117 co-expressed gene pairs were identified among bacterial keratitis DEGs and 87 pairs among fungal keratitis DEGs. In total, nine biological pathways and seven KEGG pathways were screened by analyzing DEGs in the fusional co-expression network. Conclusion: TLR4 is the representative DEG specific to bacterial keratitis, and SOD2 is the representative DEG specific to fungal keratitis, both of which are promising candidate genes to distinguish between bacterial and fungal keratitis.

Association study of single nucleotide polymorphisms in IL-10 and IL-17 genes with the severity of microbial keratitis.

PURPOSE: Exploratory analysis to assess the association of single nucleotide polymorphisms (SNPs) in the interleukin (IL) 10 and IL-17 genes with severity of contact lens keratitis. METHODS: This was a retrospective case control study of 88 contact lens keratitis cases (25 severe) and 185 healthy contact lens wearers recruited from studies conducted at Moorfields Eye Hospital and in Australia-wide during 2003-2005. Buccal swab samples were collected on Whatman FTA cards and mailed by post for DNA extraction and SNP genotyping. IL-10 (rs1800871; rs1800896; rs1800872) and IL-17 (rs1800871; rs1800896; rs1800872) SNPs were screened by pyrosequencing. Genetic association analyses were performed via Cochran-Armitage trend tests and logistic regression models using PLINK software. RESULTS: None of the SNPs tested showed evidence of association with severity of contact lens keratitis at P <  0.05. Nevertheless, minor allele G in SNP rs2397084 of the IL-17F gene was associated with increased risk of severe MK, with OR=2.1 (95% CI=0.9-4.8, P = 0.066). CONCLUSION: Our study cannot exclude with confidence that genetic variation in the IL-17 F proinflammatory cytokine is associated with more severe outcomes of MK. However, there is general body of information that the IL-17 pathway is important in the mechanisms of MK. Studies with larger power and the expanded array of laboratory tools will elucidate the exact role of IL-17 in MK.

Growth hormone-releasing hormone receptor mediates cytokine production in ciliary and iris epithelial cells during LPS-induced ocular inflammation.

The receptor for growth hormone-releasing hormone (GHRH-R) has been shown to upregulate specifically in the ciliary and iris epithelial cells and infiltrating cells in the aqueous humor in a rat model of acute anterior uveitis. Treatment with GHRHR-R antagonist alleviates significantly these inflammatory responses. Herein we investigated whether the ciliary and iris epithelial cells can respond directly to lipopolysaccharide (LPS) without the influences of circulating leukocytes to produce inflammatory mediators through a GHRH-R mediated mechanism. In explant cultures of rat ciliary body and iris, LPS caused a substantial increase of GHRH-R in 24 h. Immunohistochemistry showed a localization of TLR4, the receptor for LPS, and an elevated expression of IL-6 and IL-1ß in ciliary and iris epithelial cells after LPS treatment. LPS also elevated the level of IL-1ß, IL-6, and iNOS and increased secretion of IL-1ß and IL-6 from the explants. The GHRH-R antagonist, MIA-602, suppressed the elevated expression of IL-1ß and IL-6, and reduced the release of IL-6. Such effects were not seen for the GHRHR agonist, MR-409. When co-cultured with leukocytes, expression of GHRH-R in the ocular explants was further enhanced during LPS treatment. Our results demonstrate a direct action of LPS on ciliary and iris epithelial cells to produce pro-inflammatory factors through a GHRH-R mediated mechanism, and suggest a role of these epithelial cells, in addition to the resident antigen presenting cells, in immune surveillance of the eye. Infiltrating leukocytes may enhance these inflammatory responses by regulating GHRH-R in ciliary and iris epithelial cells, in addition to their functions of synthesizing proinflammatory cytokines.

NLRP12 promotes host resistance against Pseudomonas aeruginosa keratitis inflammatory responses through the negative regulation of NF-κB signaling.

OBJECTIVE: To investigate the role of NLRP12 in regulating Pseudomonas aeruginosa (P. aeruginosa) keratitis. MATERIALS AND METHODS: Real-Time-PCR and Western blot were performed to measure the NLRP12 level in corneas and bone marrow-derived macrophages (BMDMs) of C57BL/6 (B6) mice. B6 mice received a subconjunctival injection of lentivirus expressing active NLRP12 (NLRP12-lentivirus) or Ctl-lentivirus (as control), followed by infection of P. aeruginosa. The clinical score, slit lamp and bacterial plate count of mice were evaluated. In addition, myeloperoxidase (MPO) was detected to assess the infiltration of polymorphonuclear neutrophil (PMN). Cytokine levels were measured by Real Time-PCR and ELISA. Meanwhile, the bacterial burden was also evaluated. The activation of NF-κB signaling was determined by pIκBα/IκBα levels based on Western blot and NF-κB-dependent Luciferase activity on the basis of Luciferase assays using 293T cells. RESULTS: NLRP12 mRNA and protein levels were decreased in B6 corneas and BMDMs after P. aeruginosa infection. The over-expression of NLRP12 in B6 corneas significantly ameliorated the severity of corneal disease, bacterial burden, PMN infiltration and pro-inflammatory cytokine expression. In vitro analysis demonstrated that the up-regulation of NLRP12 suppressed pro-inflammatory cytokine production and enhanced bacterial clearance in RAW264.7 cells. The protein levels of pIκBα and IκBα were significantly decreased after NLRP12-lentivirus treatment compared with that of Ctl-lentivirus. NF-κB-dependent Luciferase activity was potently inhibited by NLRP12 infected with P. aeruginosa or cotransfected with the downstream signaling molecules including IKKα and IKKß in 293T cells. CONCLUSIONS: NLRP12 decreases the severity of P. aeruginosa keratitis, reduces corneal inflammation and bacterial burden through the down-regulation of the NF-κB signaling pathway.

Potential Role of Ocular Microbiome, Host Genotype, Tear Cytokines, and Environmental Factors in Corneal Infiltrative Events in Contact Lens Wearers.

Purpose: The purpose of this study was to explore differences in genotype, ocular surface microbiome, tear inflammatory markers, and environmental and behavioral exposures in soft contact lens (SCL) wearers with and without a history of corneal infiltrative events (CIEs). Methods: Nine SCL wearers with a recent CIE and nine age-, sex-, and SCL material- and modality-matched controls were enrolled. The Contact Lens Risk Survey, slit-lamp examination data, basal tears, conjunctival microbial cultures, and peripheral blood samples were collected. Tear inflammatory mediator concentrations, genomic DNA from swabs, and whole exome sequencing of blood samples were quantified. Results: There were no marked differences in SCL wear behaviors or exposures between case and control subjects. Predominant organisms detected among case and control subjects were Staphylococcus, Propionibacterium, Streptococcus, and Corynebacterium. Marginally higher levels of Neisseria were found in three of nine cases but zero of nine control samples (P = 0.056). A potentially deleterious missense single nucleotide polymorphism (SNP) variant in IL-6 Signal Transducer (IL6ST) was found in seven of eight cases and zero of nine controls (rs2228046; P = 0.03). The concentration of tear IL-6 was significantly higher in cases (4.5 [range, 2.1 to 6.2] pg/mL) versus controls (3.5 [range, 2.5 to 6.6] Pg/mL; = 0.02). Conclusions: Tear IL-6 concentration was higher, and SNP variants were detected in subjects with a history of CIEs compared with healthy controls. The synthesis, signaling, and ocular surface cytokine concentration of IL-6 may be related to susceptibility to CIE. A larger study population is required to further explore relationships between genetic variations, the ocular surface microbiome, inflammatory mediators, and environmental exposures.

Association between possession of ExoU and antibiotic resistance in Pseudomonas aeruginosa.

Virulent strains of Pseudomonas aeruginosa are often associated with an acquired cytotoxic protein, exoenzyme U (ExoU) that rapidly destroys the cell membranes of host cells by its phospholipase activity. Strains possessing the exoU gene are predominant in eye infections and are more resistant to antibiotics. Thus, it is essential to understand treatment options for these strains. Here, we have investigated the resistance profiles and genes associated with resistance for fluoroquinolone and beta-lactams. A total of 22 strains of P. aeruginosa from anterior eye infections, microbial keratitis (MK), and the lungs of cystic fibrosis (CF) patients were used. Based on whole genome sequencing, the prevalence of the exoU gene was 61.5% in MK isolates whereas none of the CF isolates possessed this gene. Overall, higher antibiotic resistance was observed in the isolates possessing exoU. Of the exoU strains, all except one were resistant to fluoroquinolones, 100% were resistant to beta-lactams. 75% had mutations in quinolone resistance determining regions (T81I gyrA and/or S87L parC) which correlated with fluoroquinolone resistance. In addition, exoU strains had mutations at K76Q, A110T, and V126E in ampC, Q155I and V356I in ampR and E114A, G283E, and M288R in mexR genes that are associated with higher beta-lactamase and efflux pump activities. In contrast, such mutations were not observed in the strains lacking exoU. The expression of the ampC gene increased by up to nine-fold in all eight exoU strains and the ampR was upregulated in seven exoU strains compared to PAO1. The expression of mexR gene was 1.4 to 3.6 fold lower in 75% of exoU strains. This study highlights the association between virulence traits and antibiotic resistance in pathogenic P. aeruginosa.

TLR4 modulates inflammatory gene targets in the retina during Bacillus cereus endophthalmitis.

BACKGROUND: Endophthalmitis is a serious intraocular infection that frequently results in significant inflammation and vision loss. Because current therapeutics are often unsuccessful in mitigating damaging inflammation during endophthalmitis, more rational targets are needed. Toll-like receptors (TLRs) recognize specific motifs on invading pathogens and initiate the innate inflammatory response. We reported that TLR4 contributes to the robust inflammation which is a hallmark of Bacillus cereus endophthalmitis. To identify novel, targetable host inflammatory factors in this disease, we performed microarray analysis to detect TLR4-dependent changes to the retinal transcriptome during B. cereus endophthalmitis. RESULTS: C57BL/6 J and TLR4-/- mouse eyes were infected with B. cereus and retinas were harvested at 4 h postinfection, a time representing the earliest onset of neutrophil infiltration. Genes related to acute inflammation and inflammatory cell recruitment including CXCL1 (KC), CXCL2 (MIP2-α), CXCL10 (IP-10), CCL2 (MCP1), and CCL3 (MIP1-α)) were significantly upregulated 5-fold or greater in C57BL/6 J retinas. The immune modulator IL-6, intercellular adhesion molecule ICAM1, and the inhibitor of cytokine signal transduction SOCS3 were upregulated 25-, 11-, and 10-fold, respectively, in these retinas. LIF, which is crucial for photoreceptor cell survival, was increased 6-fold. PTGS2/COX-2, which converts arachidonic acid to prostaglandin endoperoxide H2, was upregulated 9-fold. PTX3, typically produced in response to TLR engagement, was induced 15-fold. None of the aforementioned genes were upregulated in TLR4-/- retinas following B. cereus infection. CONCLUSIONS: Our results have identified a cohort of mediators driven by TLR4 that may be important in regulating pro-inflammatory and protective pathways in the retina in response to B. cereus intraocular infection. This supports the prospect that blocking the activation of TLR-based pathways might serve as alternative targets for Gram-positive and Gram-negative endophthalmitis therapies in general.

Orbital tuberculosis: Clinicopathological correlation and diagnosis using PCR in formalin-fixed tissues.

This article describes the clinicopathological correlation and challenges encountered in the diagnosis of orbital tuberculosis (TB). Retrospective, interventional case series. A chart review of six patients who underwent biopsy for an orbital mass between January 2012 and December 2013 was performed. Institutional review board approval was obtained for the study. Clinical, radiological, and laboratory findings were documented, and response to antitubercular therapy (ATT) was noted. Age at presentation ranged from 18 to 64 years and duration of complaints varied from 2 weeks to 2 months. Pain, redness, and swelling with an orbital mass on computerized tomography (CT) were the common features. None of the patients was clinically suspected to have orbital TB at presentation. History of prior contact with a patient of TB and history of prior TB were present in one case each. Chest radiography (CXR) showed pulmonary consolidation in one case. Tuberculin skin test (TST) readings ranged from nonreactive to 23 mm. The presence of sputum acid fast bacilli (AFB) was tested in three cases and was negative. Polymerase chain reaction (PCR) for MPB64 gene was positive in all six cases. All cases responded to ATT. Clinical presentation of orbital TB can be variable and can simulate conditions like inflammatory disease and malignancy. Biopsy with histopathology plays a key role in diagnosis. Supportive laboratory investigations are necessary to clinch the diagnosis in cases where histopathology is suggestive of TB. In this regard, PCR for Mycobacterium tuberculosis (MTB) on tissue biopsy specimens may be a sensitive diagnostic tool.

A dot hybridization assay for the diagnosis of bacterial keratitis.

PURPOSE: To evaluate a bacterial dot hybridization (BDH) assay for the diagnosis of bacterial keratitis (BK). METHODS: Sixty-one qualified corneal scrapings from 61 patients with suspected microbial keratitis were collected consecutively and prospectively. Among the 61 patients, 16 cases were BK and 45 cases were non-BK, including fungal keratitis, viral keratitis, parasitic keratitis, and non-microbial keratitis. Molecular diagnosis of BK in these corneal scrapes was performed using the BDH assay with three universal bacterial probes (PB1, PB2, and PB3) and three genus-specific probes (Aci, Klb, and Psu) to detect Acinetobacter, Klebsiella, and Pseudomonas, respectively. Signals were standardized after grayscale image transformation for objective validation using receiver operating characteristic (ROC) curves. RESULTS: The standardized intensities for the three universal probes differed statistically significantly between the BK group and the non-BK group. Based on the ROC curves, the sensitivities of PB1, PB2, and PB3 were 81.3%, 81.3%, and 93.8%, and the specificities were 71.1%, 88.9%, and 91.1%, respectively. The sensitivity and specificity of the Psu probe were 92% and 100%, respectively, while those of the Aci and Klb probes could not be estimated because there were no BK cases caused by Acinetobacter spp. or Klebsiella spp. CONCLUSIONS: The BDH assay is an effective molecular approach to improve the diagnosis of BK. Because the bias from bacterial contamination on the ocular surface can be minimized with signal standardization, the assay has the potential to be adopted for routine clinical practice.

miRNAs that associate with conjunctival inflammation and ocular Chlamydia trachomatis infection do not predict progressive disease.

We previously showed that conjunctival miR-147b and miR-1285 were upregulated in Gambian adults with inflammatory scarring trachoma, and miR-155 and miR-184 expression was strongly associated with conjunctival inflammation and ocular Chlamydia trachomatis infection in children from Guinea-Bissau. We investigated whether the single or combined expression of miR-147b, miR-1285, miR-155 and miR-184 was able to identify individuals with increased risk of incident or progressive scarring trachoma. Conjunctival swab samples were collected from 506 children between the ages of 4 and 12 living in northern Tanzania. These 506 samples formed the baseline sample set of a 4-year longitudinal study. Chlamydia trachomatis infection was diagnosed by droplet digital PCR and expression of miR-155, miR-184, miR-1285 and miR-147b was tested by qPCR. Individuals were assessed for incidence and progression of conjunctival scarring by comparison of conjunctival photographs taken at baseline and 4 years later. miR-184 and miR-155 were strongly associated with inflammation and infection at baseline; however, no miR was associated with 4-year scarring incidence or progression. miR-184 expression was more strongly downregulated during inflammation in non-progressors relative to progressors, suggesting that a disequilibrium in the efficiency of wound healing is a significant determinant of progressive conjunctival fibrosis.