To Örebro University

PURPOSE: To describe the clinical characteristics of exogenous episodes of endophthalmitis from which Staphylococcus epidermidis was isolated by vitreous cultures. We also explored the genomic traits of these S. epidermidis isolates and their relatedness to S. epidermidis originating from carriers and from prosthetic joint infections in the same geographical region.

METHODS: S. epidermidis isolated from cases of exogenous endophthalmitis (n = 33) were genome sequenced. Clinical features were retrospectively collected from medical records. The isolates were compared with previously sequenced S. epidermidis isolates from the nares of healthy individuals (n = 151) and from prosthetic joint infections (n = 138).

RESULTS: The most common ophthalmological procedure preceding the endophthalmitis was a posterior segment surgery (76%; 25/33), mainly intravitreal injection (70%; 23/33). These patients displayed a significantly shorter time to symptoms compared to those with an anterior segment surgery (median 3 vs. 9 days; p < 0.001), and significantly less phenotypic methicillin resistance (8%, n = 2/25 vs. 50%, n = 4/8; p = 0.02). Most isolates of S. epidermidis originating from endophthalmitis cases did not belong to known healthcare-associated lineages and did not cluster with isolates from prosthetic joint infections. Rather, they were more similar to isolates from the nares of healthy individuals.

CONCLUSIONS: Genomic data suggest that the S. epidermidis isolated from the vitreous of Swedish cases of postsurgical endophthalmitis may originate from the commensal flora of the individual, and not from the healthcare facilities. The type of preceding surgical procedure (anterior vs. posterior segment) may influence both symptom delay and the presence or absence of methicillin resistance.

Purpose: To describe the corneal microbiome in infectious keratitis in relation to contact lens wear or not, and culture outcome. To explore if targeted sequencing may provide information concerning: i) culture negative episodes, ii) variables influencing culture outcome of corneal samples dispensed in transport medium.

Methods: Prospective inclusion of patients fulfilling predefined criteria of infectious keratitis. Corneal samples were dispensed in liquid Amies medium, from which both culture and targeted sequencing of the V3-V4 region of the 16S rRNA gene were carried out. Additional standard corneal culture was also performed. Main outcome measures were bacterial findings by targeted sequencing in relation to contact lens wear and culture outcome, and identification of variables influencing corneal culture outcome of indirectly inoculated corneal samples, using quantitative data from the PCR.

Results: In all, 94 episodes of infectious keratitis were included, of which 70 (74%) had bacterial growth on corneal culture. In median, 15 (range 8-30) different bacterial genera per episode of infectious keratitis were detected by targeted sequencing. The contact lens wearers (69/94; 73%) displayed significant (p=0.01) differences in the bacterial community composition of the corneal lesion compared to non-wearers, with higher abundance of Staphylococcus spp. Corynebacterium spp. and Stenotrophomonas maltophilia. Among the culture negative episodes (n=24) Brevundimonas was found to be significantly (adjusted p<0.05) enriched. Sequencing detected a potential corneal pathogen such as Clostridium, Staphylococcus, Brevundimonas, Pseudomonas and Veillonella, with a relative abundance of at least 20% in more than half of the culture negative episodes (14/24; 58%). Bacterial density in the sample had the highest impact on culture outcome (OR 6.3; p=0.009) but also age increased the odds for a positive culture outcome (OR 1.04; p=0.034), while prior antibiotic treatment significantly reduced the odds of a positive corneal culture to a fifth (OR 0.2; p=0.031).

Conclusions: Targeted sequencing can provide a potential corneal pathogen in case of a negative culture outcome in patients with infectious keratitis, as well as providing insights on the corneal microbiome of infectious keratitis and factors influencing its composition.

Infectious keratitis is a potentially sight threatening ophthalmological emergency. Contact lens wear is a common risk factor. Diagnostic advances such as MALDI-TOF MS provides new insights into the spectrum of corneal pathogens and on microbes previously considered as commensals. Corynebacterium macginleyi was described in 1995, and in 2018, the genomic features of three isolates were reported after whole-genome sequencing. Here we describe the clinical characteristics of patients with infectious keratitis (n = 29) presumably caused by Corynebacterium macginleyi, and analyze the genomic features of C. macginleyi (n = 22) isolated from the corneal ulcers of these patients. The disease course was uneventful apart from minor interventions such as corneal cross-linking and amniotic membrane transplant. Genome sequencing and comparison revealed a highly conserved core genome of C. macginleyi. Based on the analyses of single nucleotide polymorphisms, the population could be divided into two main clades that also differed in a few clade-specific genomic islands. Patients infected with an isolate belonging to the minor clade (n = 7) presented a more severe disease. Comparisons with other corynebacterial species clearly separated C. macginleyi. C. macginleyi may be considered a corneal pathogen; genomic analysis provided insights into its population structure and disease-causing potential.

Corynebacterium macginleyi is a slow-growing, lipid-requiring bacterium that may cause ocular infections. Here, we report the complete genome sequences of two strains, T160811 and T180208, isolated from infectious keratitis. The two genomes consist of circular chromosomes of 2,431,961 bp and 2,481,998 bp, respectively, and contain high numbers of repetitive elements.

BACKGROUND: To compare two different methods of corneal culture in infectious keratitis: multiple sampling for direct inoculation and enrichment (standard method) and a single sample via transport medium for indirect inoculation (indirect inoculation method).

METHODS: Prospective inclusion of patients fulfilling predefined criteria of infectious keratitis undergoing corneal culture according to both studied methods in a randomized order.

RESULTS: = 0.002) and a significantly higher proportion of microorganisms than the indirect inoculation method, with a Cohen's kappa of 0.38 (95% CI: 0.28-0.49) for agreement between the methods. Subanalysis of culture results showed that direct inoculation on gonococcal agar only combined with the indirect inoculation method resulted in a similar rate of culture positive patients and proportion of detected microorganisms to the standard method.

CONCLUSION: Indirect inoculation of one corneal sample cannot replace direct inoculation of multiple corneal samples without loss of information. A combination of directly and indirectly inoculated samples can reduce the number of corneal samples by four without statistically significant differences in culture outcome or in the proportion of detected microorganisms.

First, to describe the microbial spectrum in infectious keratitis by both a culture and a targeted sequencing approach; second, to describe the clinical and genetic characteristics of Corynebacterium macginleyi; and third, to study different aspects of the corneal culture process by comparing two instruments used for sampling and two sampling and inoculation strategies. 

The five studies in this thesis made use of two retrospective study populations and one prospective study population.

In the retrospective population the microbial spectrum by corneal culture was explored, Gram-positive bacteria, mainly coagulase-negative staphylococci, Staphylococcus aureus, and Corynebacterium spp. were commonly isolated. In the prospective study population, culture detected 13 different bacterial genera, while targeted sequencing identified a total of 168 different bacterial genera, with individual samples having a median of 13 (7–28) genera. Culture and sequencing showed an 82% agreement on the bacterial genera detected by culture. Genome sequencing and analysis of C. macginleyi revealed two different clades of which the minor clade (n=7), not previously described, exhibited a more complicated disease course. Cotton tipped applicators generated a significantly higher rate (44%) of positive corneal cultures on solid media than knife blades (31%). Direct transferal of multiple corneal samples to culture media generated a significantly higher rate of positive corneal cultures (61%) than indirect transferal through a single transport medium (44%).

In conclusion, the microbial spectrum in a Swedish population is similar to previously described in Europe, and C. macginleyi may be considered a corneal pathogen. Targeted sequencing may gain clinical application if further developed. The findings of this thesis indicate that a cotton tipped applicator may be sufficient for corneal sampling for direct inoculation, and the corneal culture procedure can be simplified from seven samplings on four different media to three samplings on two media.

Objective: This study aimed to compare the efficacy of a cotton tipped applicator and a knife blade in obtaining corneal samples in patients with infectious keratitis.

Methods and analysis: This is a retrospective cohort study of patients with suspected infectious keratitis during 2004-2014. Samples for corneal culture were obtained by a cotton tipped applicator and a knife blade, and directly inoculated on GC agar, blood agar and Sabouraud agar.

Results: In all, 355 patients were included. Corneal sampling by cotton tipped applicator yielded a significantly higher rate of patients with positive corneal culture, 156/355 (43.9%), compared with knife blade, 111/355 (31.3%) (p<0.001). On a patient level, the culture results obtained by the cotton tipped applicator and the knife blade were identical in 269/355 (76%) of the patients. The overall agreement between the two instruments on microbial level was 0.66 (Cohen's kappa 95% CI 0.60 to 0.72).

Conclusion: Corneal sampling by cotton tipped applicator generated a higher rate of positive corneal cultures and a higher proportion of isolated microbes than by knife blade. Future studies with randomised sampling order are needed to establish which instrument, cotton tipped applicator or knife blade, is the most effective in sampling microbes for direct inoculation in patients with infectious keratitis.

PURPOSE: To describe predisposing risk factors, causative microorganisms and their antibiotic susceptibility patterns in infectious keratitis during an 11-year period in Region Örebro County, Sweden.

METHODS: This is a descriptive study conducted as a retrospective audit of clinical records. Patients who received treatment for infectious keratitis at any of the three ophthalmological departments within Region Örebro County, Sweden, between 2004 and 2014 were included if they fulfilled the predefined criteria for infectious keratitis. Data regarding culture results, antibiotic susceptibility pattern and risk factors for infectious keratitis were obtained from medical records and microbiological reports.

RESULTS: In total, 398 episodes of infectious keratitis in 392 patients were included, and 285 were culture positive. The most common predisposing risk factor was contact lens wear (45%). Coagulase-negative staphylococci (39.6%) was the most commonly isolated type of organism. Staphylococcus aureus (15.1%) followed by Moraxella spp. (7.4%) and Pseudomonas aeruginosa (6.7%) were among the most common isolated bacteria not considered to be commensal. Reduced susceptibility to fluoroquinolones was observed in five of 43 S. aureus isolates and in four of nine Streptococcus pneumoniae isolates.

CONCLUSION: The most common predisposing risk factor for keratitis was contact lens wear. Among the most common microbes, not considered to be exclusively commensals, isolated from the cornea in microbial keratitis were S. aureus, Moraxella spp. and P. aeruginosa. The antibiotic susceptibility patterns showed low proportion of resistance. Empiric treatment of suspected infectious keratitis with topical fluoroquinolones and chloramphenicol might be considered in a setting like ours pending culture results.