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.

Epidemiology, risk factors, and clinical outcomes in severe microbial keratitis in South India.

PURPOSE: Here, we report risk factors associated with outcome in severe bacterial keratitis (BK), fungal keratitis (FK), and Acanthamoeba keratitis (AK) in India. METHODS: Prospective observational cohort study conducted in Aravind Eye Hospital, India. Adults presenting with severe microbial keratitis (MK) were enrolled (size ≥3 mm) and followed to 21 days post-enrolment. Ulcer clinical features were recorded at presentation. Outcomes by final visit were classified as good (completely healed or reduced infiltrate size) or poor (enlarged infiltrate size, perforated, or surgery performed). RESULTS: Of 252 participants with severe MK, 191 had FK, 18 had AK, 19 had BK, 4 had mixed BK/FK, and 20 were microbiologically negative. Median age was 50 years (interquartile range [IQR]: 37-60 years), 64% were male, 63% were agriculturalists, and 45% had no formal education. Corneal trauma occurred in 72%, and median symptom duration before presentation was 7 days (IQR: 5-15 days). Clinical features associated with FK were feathery margins (p < 0.001), raised profile (p = 0.039), or dry surface (p = 0.007). Hypopyon was more likely in BK (p = 0.001) and ring infiltrate in AK (p < 0.001). Ulcers with poor outcome (n = 106/214) were more likely to be larger (odds ratio [OR]: 1.63, 95% confidence interval [CI]: 1.30-2.05, p < 0.001), involve the posterior cornea at presentation (OR: 2.31, 95% CI: 1.16-4.59, p = 0.017), involve Aspergillus sp. (OR: 3.23, 95% CI: 1.26-8.25, p = 0.014), or occur in females (OR: 2.04, 95% CI: 1.03-4.04, p = 0.04). Even after treatment, 34% (n = 76/221) had severe visual impairment by the final visit. CONCLUSIONS: Severe MK occurred predominantly in agriculturalists post-corneal trauma and often had poor outcomes. Provision of community-based eyecare may allow earlier treatment and improve outcomes.

In Vitro Evaluation of the Drug Reservoir Function of Human Amniotic Membrane Using Moxifloxacin as a Model Drug.

PURPOSE: To evaluate the in vitro, extended drug reservoir function of human amniotic membrane (HAM) of different thicknesses impregnated with moxifloxacin. METHODS: HAM buttons (12 mm) were soaked with freshly prepared 0.5% wt/vol topical moxifloxacin at different soaking time intervals: 3 hours (group I), 6 hours (group II), 12 hours (group III), 24 hours (group IV), and 48 hours (group V). They were then transferred into 1 mL of fresh simulated tear fluid (pH-7.4) and incubated at 37°C. The release kinetics of moxifloxacin was studied by analyzing the amount of drug in simulated tear fluid collected at different time intervals from each pretreated HAM for 3 weeks. In another experiment, thin and thick HAMs were selected based on weight and soaked with moxifloxacin for 24 hours, and the release kinetics was studied for 7 weeks. All samples were stored at -80°C until analysis by high-performance liquid chromatography. RESULTS: No significant difference was observed between different soaking times and the release of moxifloxacin. The cumulative amount of moxifloxacin released from thick HAM was found to be statistically significant compared with thin HAM (P < 0.05). CONCLUSIONS: Our in vitro data showed that the sustained release of moxifloxacin from HAM was achieved up to 7 weeks. The entrapment efficiency of moxifloxacin was significantly higher in thicker HAM than in thin HAM. Moxifloxacin-impregnated HAM application can be considered in bacterial keratitis to provide sustained drug delivery through a biological bandage system for up to a period of 7 weeks.

Data set for the mass spectrometry based exoproteome analysis of Aspergillus flavus isolates.

Aspergillus flavus is one of the predominant causative organisms of mycotic keratitis in tropical parts of the world. Extracellular proteins are the earliest proteins that come in contact with the host and have a role in the infection process. Exoproteins of A. flavus isolated from infected cornea, sputum and a saprophyte were pooled and identified using high resolution mass spectrometry in order to get the total exoproteome from cultures isolated from different sources. A total of 637 proteins was identified from the pooled A. flavus exoproteome. Analysis based on GO annotations of the 637 identified proteins revealed that hydrolases form the predominant class of proteins in the exoproteome. Interestingly, a greater proportion of the exoproteins seem to be secreted through the non-classical pathways. This data represent the first in-depth analysis of the representative A. flavus exoproteome of a large set of isolates from distinct sources. This data have been deposited to the ProteomeXchange with identifier PXD001296.

Pathogen induced changes in the protein profile of human tears from Fusarium keratitis patients.

Fusarium is the major causative agent of fungal infections leading to corneal ulcer (keratitis) in Southern India and other tropical countries. Keratitis caused by Fusarium is a difficult disease to treat unless antifungal therapy is initiated during the early stages of infection. In this study tear proteins were prepared from keratitis patients classified based on the duration of infection. Among the patients recruited, early infection (n = 35), intermediate (n = 20), late (n = 11), samples from five patients in each group were pooled for analysis. Control samples were a pool of samples from 20 patients. Proteins were separated on difference gel electrophoresis (DIGE) and the differentially expressed proteins were quantified using DeCyder software analysis. The following differentially expressed proteins namely alpha-1-antitrypsin, haptoglobin α2 chain, zinc-alpha-2-glycoprotein, apolipoprotein, albumin, haptoglobin precursor - ß chain, lactoferrin, lacrimal lipocalin precursor, cystatin SA III precursor, lacritin precursor were identified using mass spectrometry. Variation in the expression level of some of the proteins was confirmed using western blot analysis. This is the first report to show stage specific tear protein profile in fungal keratitis patients. Validation of this data using a much larger sample set could lead to clinical application of these findings.