Ozkan Jerome, Coroneo Minas, Sandbach Jennifer, Subedi Dinesh, Willcox Mark, Thomas Torsten
School of Optometry and Vision Science, University of New South Wales, Sydney, Australia; School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, Australia.
Department of Ophthalmology, Prince of Wales Hospital, University of New South Wales, Sydney, Australia.
Ocul Surf. 2021 Jan;19:169-175. doi: 10.1016/j.jtos.2020.05.010. Epub 2020 Jun 1.
The ocular surface microbiota are recognised as one of causative microorganisms in post-procedural endophthalmitis but in many cases the vitreous tap is culture negative. This study investigated bacterial contamination of intravitreal (IVT) needles using multiple approaches covering culturing, 16S rRNA gene sequencing, fluorescent in situ hybridisation (FISH) and scanning electron microscopy (SEM).
IVT needles were obtained immediately after injection from patients undergoing treatment for predominantly age-related macular degeneration. Eighteen needles were analysed by culturing on chocolate blood agar. In addition, 40 needles were analysed by extracting DNA and paired-end sequencing of the 16S rRNA gene. Sequences were quality filtered (USEARCH), taxonomically classified (SILVA) and contaminant filtered (DECONTAM). Nine needles were analysed by either FISH using the bacterial probe EUB338 or SEM.
Using culturing, three bacteria were identified from 5 of 18 needles (28%) - Kocuria kristinae, Staphylococcus hominis and Sphingomonas paucimobilis. The negative control needles showed no growth. Following rigorous data filtering, bacterial community analysis using 16S rRNA gene sequencing showed the presence of predominantly Corynebacterium but also Pseudomonas, Acinetobacter, Sphingomonas, Staphylococcus and Bacillus on the needles. Cocci-shaped cells in a tetrad formation were observed using FISH, while SEM images showed cocci-shaped bacteria in pairs and irregular tetrads.
The study showed evidence for a large diversity of bacteria on IVT needles and visually confirmed their adherence. The diversity was similar to that found on the ocular surface and in conjunctival tissue. This suggests the risk of exogenous endophthalmitis remains even with sterilization of the conjunctival surface.
眼表微生物群被认为是术后眼内炎的致病微生物之一，但在许多情况下，玻璃体穿刺液培养呈阴性。本研究采用多种方法，包括培养、16S rRNA基因测序、荧光原位杂交（FISH）和扫描电子显微镜（SEM），对玻璃体内（IVT）注射针的细菌污染情况进行了调查。
从主要接受年龄相关性黄斑变性治疗的患者注射后立即获取IVT针。18根针在巧克力血琼脂上进行培养分析。此外，40根针通过提取DNA并对16S rRNA基因进行双端测序进行分析。序列经过质量过滤（USEARCH）、分类学分类（SILVA）和污染物过滤（DECONTAM）。9根针通过使用细菌探针EUB338的FISH或SEM进行分析。
通过培养，从18根针中的5根（28%）鉴定出三种细菌——科氏葡萄球菌、人葡萄球菌和少动鞘氨醇单胞菌。阴性对照针没有生长。经过严格的数据过滤后，使用16S rRNA基因测序进行的细菌群落分析显示，针上主要存在棒状杆菌，但也有假单胞菌、不动杆菌、鞘氨醇单胞菌、葡萄球菌和芽孢杆菌。使用FISH观察到呈四联排列的球菌形细胞，而SEM图像显示成对和不规则四联的球菌形细菌。
该研究表明IVT针上存在多种细菌，并通过视觉确认了它们的附着。这种多样性与眼表和结膜组织中发现的相似。这表明即使结膜表面经过消毒，外源性眼内炎的风险仍然存在。
