Purpose

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).

Methods

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.

Results

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.

Conclusions

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.

中文翻译：

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眼表微生物组对玻璃体内针的细菌污染。

目的

在手术后眼内炎中，眼表微生物群被认为是病原微生物之一，但在许多情况下，玻璃体水龙头培养阴性。这项研究使用多种方法研究了玻璃体内（IVT）针的细菌污染，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）。

方法

注射后立即从主要患有年龄相关性黄斑变性的患者中获得IVT针。通过在巧克力血琼脂上培养来分析十八个针。此外，通过提取DNA和16S rRNA基因的配对末端测序分析了40根针。序列经过质量过滤（USEARCH），分类分类（SILVA）和污染物过滤（DECONTAM）。使用细菌探针EUB338或SEM通过FISH分析九个针头。

结果

-使用培养，三种细菌是从5 18的针状物（28％）鉴定Kocuria kristinae，人葡萄球菌和鞘氨醇单胞菌。阴性对照针无生长。经过严格的数据过滤后，使用16S rRNA基因测序的细菌群落分析显示，在针上主要存在棒杆菌，还存在假单胞菌，不动杆菌，鞘氨醇单胞菌，葡萄球菌和芽孢杆菌。使用FISH观察到四分体形成的球菌形细胞，而SEM图像显示成对和不规则四分体的球菌形细菌。

结论

该研究显示了IVT针上细菌种类繁多的证据，并在视觉上证实了它们的粘附性。该多样性类似于在眼表和结膜组织中发现的多样性。这表明即使对结膜表面进行消毒，仍存在外源性眼内炎的风险。