Prevalence and Molecular Characterization of Metallo ß-Lactamase Producing Gram-Negative Pathogens Causing Eye Infections.

Purpose: Metallo ß-lactamases (MßL) production is a worldwide problem, particularly in gram-negative bacteria. As scanty data is available on the prevalence of MBL, the present study is being undertaken to determine the prevalence, antibacterial sensitivity patterns, and molecular characterization of MßL associated resistant genes in gram-negative bacteria isolated from ocular infections. Material and Methods: At a tertiary eye care center in south India, 359 gram-negative pathogens, 200 isolates from eye infections, and 159 isolates from normal flora of the eye were studied. A gold standard microbiology method was used to identify the isolates. An antibiotic double disc synergy test and a combination disc test were used to detect MßL production. Multiplex PCR was used to investigate the molecular characteristics of the MßL encoding genes blaVIM, blaIMP, and blaNDM. Results: Of the 359 gram-negative bacterial pathogens, Pseudomonas aeruginosa 108 (30.1%) and Enterobacter agglomerans 46 (12.8%) were commonly isolated. High prevalence of P. aeruginosa 81% (17 strains) was detected as an MßL producer and it shows 100% resistance to 2nd and 3rd generation cephalosporins and meropenem. Multiplex PCR detected only the blaVIM gene in 56 (28%) of various eye infections and 27 (17%) of normal flora of the gram-negative bacteria (GNB). The blaVIM gene is detected predominantly in 51.8% of keratitis and 21.4% of postoperative endophthalmitis. High prevalence of the gene was detected in P. aeruginosa 42.9% (24 of 56) and Alcaligens denitrificans 10.7% (6 of 56) from eye infections. Whereas, in the control group, P. aeruginosa and E. coli each had 14.8% (4 of 27) that were shown positive. Conclusion: The emerging MßLs mediated resistance among P. aeruginosa is a challenging task for ophthalmologists, especially in patients with endophthalmitis and bacterial keratitis. This local knowledge will aid in advising appropriate antibiotic use and avoiding unnecessary antibiotic prescriptions, which are highly warranted.

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.

Periconia species - An unusual fungal pathogen causing mycotic keratitis.

PURPOSE: The purpose of this study is to determine the epidemiology, risk factors, clinical features, and treatment outcome of molecularly diagnosed Periconia keratitis. METHODS: Clinical records of all culture proven fungal ulcers with molecular identification suggestive of Periconia species who presented to a single tertiary referral center from January 2012 to December 2013 were retrospectively analysed. RESULTS: Among 1356 cases of keratomycosis, 8 (0.6%) patients were affected due to Periconia species. The mean age of presentation was 59 years with males (n = 6; 75%) were more commonly affected than females (n = 2; 25%). Significant history of trauma was present only in one patient. The infiltrate size was less than 5 mm in majority of patients 75% (n = 6). 50% (n = 4) responded to antifungal, 12.5% (n = 1) responded to antibacterial, 12.5% (n = 1) required therapeutic penetrating keratoplasty, 25% (n = 2) lost to follow up after first visit. The mean duration of treatment in healed cases was 20 days. CONCLUSION: This is the first report on Periconia sp causing human corneal ulcer. This study signifies the importance of molecular identification in the diagnosis of rare fungi which will improve our understanding on disease pathology and outcome. Visual prognosis appears good if the infection is diagnosed and topical antifungal interventions started early.

Clinical and demographic study of non-tuberculous mycobacterial ocular infections in South India.

PURPOSE: To describe demographics, risk factors, antibiotic susceptibility, management and outcomes of ocular infections caused by non-tuberculous mycobacteria (NTM). METHODS: A retrospective review of medical case records and microbiology records of patients with ocular infections that were culture positive for non-tuberculous Mycobacteria from January 2014 to December 2018 was done. Antibiotic susceptibility profile was done based on the CLSI guidelines. Laboratory diagnosis for the NTM Species was done by conventional microbiological methods. The species identification was done for stored isolated utilizing polymerase chain reaction targeting 16S rDNA and rpoB gene, followed by DNA sequencing and phylogenetic analysis. RESULTS: Twenty patients with NTM ocular infections were identified during the study period. A majority of cases presented as 12 infectious keratitis (60%) and three suture-related corneal infiltrates (15%). Common risk factors were history of trauma in 9 (45%) patients and history of ocular surgery in 5 (25%) patients. Patients were treated with combination of amikacin and flouroquinolones/chloramphenicol (70%) and surgical interventions were performed in 25% cases. Only twelve isolates were stored and ten isolates were identified as the M. abscessus subsp. abscessus and two isolates as M. abscessus subsp. massiliense by sequencing and phylogenetic analysis. Majority of the NTM were sensitive to amikacin (75%) followed by moxifloxacin, ciprofloxacin, cephotaxime and tobramycin (35%). CONCLUSION: High degree of clinical suspicion, multidrug antibiotic therapy and timely surgical intervention in patients with NTM infections, are advised for better clinical outcomes. Prior ocular trauma, prior ocular surgery and presence of biomaterials were the major predisposing factors. Earlier surgical intervention in cases where abscesses or biomaterials are involved, is necessary for rapid recovery.

Molecular identification of Nocardia species causing endophthalmitis using multilocus sequence analysis (MLSA): a 10-year perspective.

Introduction. Nocardia spp. can cause several ocular infections, such as keratitis, endophthalmitis and scleral abscesses. Molecular identification of Nocardia spp. by 16S rDNA sequencing is the gold standard method at present for species identification, but closely related species can only be identified by multilocus sequence analysis (MLSA) of housekeeping genes.Aim. The major objective was to profile Nocardia species, antibiotic susceptibility patterns and clinical outcomes in endophthalmitis patients.Methodology. Between January 2009 and December 2018, endophthalmitis patients who were diagnosed with Nocardia infection based on microscopic and culture characteristics were selected. Antibacterial susceptibility tests were performed and Nocardia speciation was performed using MLSA and phylogenetic tree analysis of the 16 s rRNA gene and the gyrB, hsp65 and secA1 genes.Results. A total of 43 culture-proven patients were identified during the study period. All isolates were 100 % sensitive to amikacin and 98 % resistant to ceftazidime. Fluoroquinolone sensitivity was observed in the range of 58 to 72 %. Year-wise analysis of antibiotic resistance patterns revealed there was a significant increase in resistance to fluoroquinolones. Twenty-two isolates were stored and six different species were identified. Nocardia farcinica (n=10) was found to be the most predominant, followed by Nocardia cyriacigeorgica (n=4), Nocardia otitidiscaviarum (n=3), Nocardia amikacinitolerans (n=2), Nocardia puris (n=2) and Nocardia higoensis (n=1).Conclusions. N. farcinica is the major pathogen, and this is the first report to identify N. otitidiscaviarum, N. amikacinitolerans and N. higoensis as causing endophthalmitis. Overall, visual outcomes were mostly poor even after aggressive management.

Genotyping and phylogenetic analysis of Pythium insidiosum causing human corneal ulcer.

Pythium insidiosum belongs to the Oomycetes, which are known to cause serious life-threatening infectious condition in humans and animals. Corneal infections caused by P. insidiosum are rare and difficult to treat. The molecular-based diagnosis of Pythium is employed for the species identification and to study molecular phylogenetic relationship. Based on Cytochrome oxidase II (cox II) gene, P. insidiosum is categorized into three clades or groups: Clade-I or ATH (American strains), Clade-II or BTH (American, Asian, and Australian strains), and Clade-III or CTH (mostly Thailand strains). This study focused on the molecular identification of Pythium insidiosum from patients with corneal ulcer using ITS regions and clade identification by cox II gene sequencing and correlated with the clinical outcome. The isolates were collected from Aravind Eye Hospital, Madurai, India, from April to December 2018. Through the microbiological laboratory reports, 15 isolates of Pythium sp. from keratitis patient were selected, followed by DNA extraction, ITS, and cox II gene sequencing and phylogenetic analysis using the reference sequences from NCBI database. All 15 P. insidiosum isolates were phylogenetically clustered together as a single group and where also placed distantly from other Pythium species (outgroup). Most ocular isolates fell into either clade BTH or clade CTH, and none of our ocular isolates were in clade ATH. Two of the strains were very distinct and did not match any of the clusters indicating different lineages. There was no significant difference between clinical outcome and genotype of P. insidiosum.

Clinical presentations, genotypic diversity and phylogenetic analysis of Acanthamoeba species causing keratitis.

Introduction. Acanthamoeba keratitis is a sight-threatening corneal infection that is commonly reported among contact lens users and those suffering from corneal trauma. The prevalence of Acanthamoeba species or genotypes in causing keratitis infection is not well known.Aim. This study was conducted to identify and genotype Acanthamoeba isolates from keratitis patients, targeting the ribosomal nuclear subunit (Rns) region, and describe the associated clinical presentation and treatment outcome.Methodology. Thirty culture-confirmed patients with Acanthamoeba keratitis, identified in a tertiary eye care centre in South India during the period from December 2016 to December 2018, were included in this study. The data collected from patient records include demographic details, history of illness, mode of trauma, treatment history and follow-up status. The genotype and species were identified based on the Rns sequence and phylogenetic tree analysis.Results. Acanthamoeba culbertsoni was the most predominant keratitis-causing species, followed by Acanthamoeba quina, Acanthamoeba castellanii, Acanthamoeba healyi, Acanthamoeba hatchetti, Acanthamoeba polyphaga and Acanthamoeba stevensoni. Three major genotypes were identified (T4, T11 and T12), with the T4 genotype being the most predominant, with four subclusters, i.e. T4A, T4B, T4D and T4E. This is the first report on corneal infection by the A. stevensoni T11 genotype and the A. healyi T12 genotype. No significant correlation was observed between the clinical outcomes of corneal disease and the genotypes or species.Conclusion. Rns genotyping is very effective in identifying the Acanthamoeba species and genotype in keratitis. Genotyping of Acanthamoeba spp. will help to advance our understanding of genotype-specific pathogenesis and geographical distribution.