In Vitro Susceptibility to Different Topical Ophthalmic Antibiotics of Bacterial Isolates from Patients with Conjunctivitis

Purpose: To test in vitro the susceptibility to different commercially-available topical antibiotics of isolates from patients with conjunctivitis, evaluated at the Microbiology Service of Turin Ophthalmic Hospital between January 2005-February 2007.

Methods: Bacterial isolates were cultured using both liquid and solid media. The four most frequent isolates were analyzed and their in vitro susceptibility to levofloxacin, ofloxacin, norfloxacin, lomefloxacin, tobramycin, netilmycin, ampicillin, and chloramphenicol was tested, using the Kirby-Bauer diffusion method and National Committee for Clinical Laboratory Standards (NCCLS) serum standards.

Results: The four most common bacteria isolates were: Staphylococcus aureus (27%), viridians Streptococci (22%), Staphylococcus epidermidis (16%) and streptococcus pneumoniae (13%). The in vitro susceptibility to levofloxacin was highest (p<0.01) for all bacterial isolates.

Conclusions: In vitro susceptibility tests showed that levofloxacin had the highest cumulative efficacy against bacterial isolates. Netilmycin showed the highest efficacy against staphylococcus aureus, the most common bacterial isolate in this study.     

人、兔角膜上皮在体外培养中的形态特征

应用组织培养技术对人、兔角膜上皮进行了培养,并对其形态及超微结构进行了观察。结果表明:培养的人、兔角膜上皮均呈不规则的多角形镶嵌排列,具有膜状生长的特点。细胞核大,圆或椭圆,有1～3个核仁。细胞骨架染色显示蓝色纤维状结构在细胞内的不同分布。扫描电镜可见表面微绒毛较多的暗细胞及较少的明细胞。透射电镜显出上皮除原有的细胞器外,还表现出核仁、张力丝、糖原、次级溶酶体、指状交叉等增多。     

人眼小梁细胞体外培养及生物学特性研究

目的:建立人眼小梁细胞培养方法并研究其生物学特性。方法:以体外组织块培养的方法获得培养的人小梁细胞,应用光镜、电镜观察细胞的形态学特征,并观察其免疫组化特性和细胞的生长曲线。结果:光镜下小梁细胞为扁平多角形、单层生长;电镜下细胞连接为点粘连和缝隙连接、细胞表面可见微绒毛、胞浆细胞器丰富;免疫组化染色对抗纤维连接蛋白(Anti—FN)、抗层粘连蛋白(Anti—LN)、抗神经元特异性烯醇化酶(Anti—NSE)单抗呈阳性,对抗第Ⅷ因子(Anti-ⅧFactor)单抗呈阴性;传代小梁细胞繁殖时间较长,10天后为平台期。结论:根据体外培养的小梁细胞的形态特点、生长特征、免疫组化特性可对其进行鉴定。人小梁细胞体外培养的成功,为在细胞和分子水平研究青光眼的发病机制提供了有利的条件。眼科学报 1996;12:64—69。     

增殖性玻璃体视网膜病变视网膜前膜体外培养及免疫组化鉴定

目的 探讨视网膜前膜组织培养中存在的细胞迁出率低、传代困难等问题;观察培养细胞的成分及体外培养细胞迁出率和增殖力与PVR发病时间的相关性.方法 使用组织块培养法对手术中剥离的PVR患者视网膜前膜组织进行体外培养;双目倒置显微镜下观察细胞生长状况;DAB显色法对细胞进行免疫组化鉴定.结果 PVR在3个月内的细胞迁出率与3个月以上的细胞迁出率和细胞传代率都有统计学意义;共培养31例前膜组织其中23例有细胞迁出,14例成功进行了细胞传代,8例传至第四代;免疫组化鉴定见:s-100、CK为阳性,Vimt、GFAP仅少部分细胞呈阳性,CD34呈阴性反应.结论 PVR 3个月内的患者的视网膜前膜组织是进行视网膜前膜组织体外培养的最佳选材;培养的细胞视网膜色素上皮细胞为主,含有少部分成纤维细胞及神经胶质细胞.     

离体及活体念珠菌在共聚焦显微镜下的特征

目的：应用活体激光共聚焦显微镜（IVCM）观察离体培养及活体状态下念珠菌孢子及假菌丝的典型 特征,为念珠菌性角膜炎的早期诊断提供依据。方法：比对观察研究。收集2016年1月到2019年12月 在山东第一医科大学附属眼科医院被诊断为念珠菌性角膜炎的26例（26眼）患者资料,同时取白色 念珠菌和近平滑念珠菌进行实验室真菌培养,应用IVCM分别观察离体及活体状态下念珠菌孢子及 假菌丝的典型形态。结果：离体与活体状态下念珠菌孢子均呈排列均匀的点状结构,形状类圆形, 直径约2～5 μm,边界清晰;假菌丝呈高反光线状结构。活体状态下孢子反光明显增强,18眼呈分散 排列点状强反光结构,11眼呈团雾状中强反光结构;假菌丝形态变异较大,2眼菌丝形体细长,5眼 呈芽孢结构,可见分支,1眼呈高对比度的不规则细长短棒状,1眼可同时观察到不规则细长短棒状 和芽孢结构。白色念珠菌假菌丝结构较细长,长度约20～150 μm,直径约2～10 μm,与母细胞连接 处收缩呈缢痕;近平滑念珠菌假菌丝短粗,长度约30～100 μm,直径2～5 μm,部分可见高亮的分支 芽孢结构。结论：念珠菌性角膜炎在IVCM下呈现典型的孢子和假菌丝结构,离体和活体状态下孢 子形态较为一致,而假菌丝形态变异较大,IVCM可用于念珠菌性角膜炎的早期诊断。     

A Study of the Characteristics of Candida with In Vitro and In Vivo Confocal Microscopy

Objective: To use in vivo laser confocal microscopy (IVCM) to observe the in vitro culture and living characteristics of candida spores and pseudohyphaes, and to provide abasis for the early diagnosis of candida keratitis. Methods: The data of 26 patients (26 eyes) who were diagnosed with candida keratitis in the Eye Hospital of Shandong First Medical University from January 2016 to December 2019 wascollected. At the same time, candida albicans and candida parapsilosis were taken for laboratory fungal culture, and the typical morphology of candida spores and pseudohyphae in vitro and in vivo were observed by IVCM. Results: Candidaspores in vitro and in vivo presented uniformly in the shape of round spots with a diameter of 2-5 μm and a clear boundary. Pseudohyphae have a highly reflective linear structure. The reflection of spores was significantly enhanced in vivo. Eighteen eyes showed a scattered arrangement of spot-like, strongly reflective structure, and 11 eyes showed a dense fog structure. The morphology of pseudomycelia varied greatly. The mycelia in 2 eyes were slender, the spore structure was present in 5 eyes, and branches were visible. In 1 eye, an irregular thin and long rod shape with high contrast was observed, and in another eye, an irregular thin and long rod shape and spore structure could be observed simultaneously. Candida albicans pseudohyphae were relatively slender, with a length of 20-150 μm and a diameter of 2-10 μm. Pseudohyphae of candida parapsilosis were short and thick, with a length of about 30-100 μm and a diameter of 2-5 μm. Part of the branch spore structure was highlighted. Conclusions: Candida keratitis showstypical spore and pseudohyphae structures with IVCM. The morphology of candida keratitis spores in vitro and in vivo are consistent with observations under IVCM, but the morphology of pseudohyphaes are relatively different. IVCM can be used for the early diagnosis of candida keratitis.     

In vitro differentiation of cells of the lens epithelium of human fetus

Cells dissociated from fetal human lens epithelium of approximately 9 and 15 weeks after conception were cultured as monolayers in vitro. About 25 days after inoculation, a number of transparent piles of highly swollen cells appeared on the epithelial cell sheet. Ultra-structurally, these piles are assemblages of elongated cells with typical profiles of mature lens fibers. Gamma-crystallin which is undetectable in the lens epithelium is immunologically detected in late stage cultures containing these piles. Thus, it is concluded that cells of the human fetal lens epithelium can differentiate into lens fibers in monolayer culture.     

Phase-transition W/O Microemulsions for Ocular Delivery: Evaluation of Antibacterial Activity in the Treatment of Bacterial Keratitis

Purpose: Moxifloxacin (MXN) is widely prescribed for the treatment of bacterial keratitis. The conventional MXN solution has several limitations, including short precorneal residence time and poor intrastromal bioavailability, requiring frequent instillation of the drug to achieve the desired therapeutic effect. To circumvent this problem, the W/O (water-in-oil) microemulsion (ME) system was utilized for sustained release and improved precorneal retention.

Methods: The pseudo-ternary phase diagrams were developed and various MEs were prepared using two non-ionic surfactants, Tween 80 and Span 20, with isopropyl myristate and acetate buffer. Physicochemical parameters, in vitro drug release and in vitro antibacterial activity were studied. The in vivo antimicrobial efficacy of optimized microemulsion (ME 10) was studied in an experiment on bacterial keratitis in rabbit eyes and compared with that of the marketed eye drops.

Results: The optimized microemulsion (ME 10) displays as an average globule size of <40 nm. The developed MEs showed acceptable physico-chemical behaviour, good stability for 3 months and exhibited sustained drug release. Greater efficacy in experimental bacterial keratitis in rabbit eyes was also observed in comparison with marketed drug solution.

Conclusions: The developed MEs are a viable alternative to conventional eye drops, because of its ability to enhance bioavailability through its longer precorneal residence time and its ability to sustain the release of the drug.     

In vitro and comparative study on the extracellular enzyme activity of molds isolated from keratomycosis and soil

AIM:To isolate and identify the molds involved in mycotic keratitis; to isolate corresponding species from soil samples; to compare the extracellular enzyme activity indices of the molds isolated from keratitis cases and the corresponding soil isolates.METHODS:The specimens were collected from the target patients attending the microbiology laboratory of tertiary eye hospital in Coimbatore, Tamilnadu state, India. The isolates were subjected for identification based on the growth on solid media, direct microscopy and lacto phenol cotton blue wet mount preparation. Extracellular enzymes such as lipase, deoxyribonuclease (DNase), α-amylase, protease, cellulase and pectinase produced by the fungalisolates were screened on solid media supplemented with the corresponding substrates. Based on growth and zone diameter, the enzyme activity indices were calculated and were compared with that of the soil fungalisolates.RESULTS:A total of 108 clinical samples were collected from a tertiary eye care hospital and out of which 60 fungal isolates were obtained. Among these, Fusarium spp. (n=30), non sporulating molds (n=9), Aspergillus flavus (n=6), Bipolaris spp. (n=6), Exserohilum spp. (n=4), Curvularia spp. (n=3), Alternaria spp. (n=1) and Exophiala spp. (n=1)were identified and designated as FS1-30, NSM1-9, AF1-6, BS1-6, ES1-4, CS1-3, AS1 and EX1, respectively. For comparative analysis, soil samples were also collected from which, one isolate of each Fusarium spp., Aspergillus flavus, Bipolaris spp., Exserohilum spp., and Curvularia spp., respectively were selected. Highest lipase activity was seen in corneal isolate NSM2 (EAI= 2.14). The DNase activity was higher in NSM9 (EAI=1.88). In case of protease, Fusarium spp. (FS9) had prominent enzyme activity index of 1.38; α-amylase activity was also superior in corneal isolate FS13 with EAI of 1.63 when compared to other isolates. The enzyme activity index for cellulase was also noted to be higher in corneal isolates i.e. NSM7 with EAI of 1.98 when compared to other corneal and soil isolates. The pectinase activity index was also prominent for corneal isolate NSM5 versus the soil isolates, SAF1, SFS1, SES1, SBS1 and SCS 1 as 1.76 versus 1.47, 1.38, 1.16, 1.11 and 1.14, respectively.CONCLUSION: The most common isolate was Fusarium spp. followed by Aspergillus, Curvularia, Exserohilum, Bipolaris, Exophiala and Alternaria species. Enzyme activity indices (EAI) of the enzymes analysed varied with the clinical and soil isolates with respect to protease and cellulase (P=0.01). Of all the strains compared it was noted that mean EAI was greater in many clinical fusarial isolates followed by non sporulating molds.     

Human Retinal Vessels in Tissue Culture: A Preliminary Report of the Effect of Acute Glucose Poisoning on Cultured Vascular Cells

To delineate the factors involved in the pathogenesis of human retinal vasculopathies, an in vitro model of human retinal vascular cells was developed by using cadaver eyes enucleated four to eight hours after death and stored at +4 C for three to seven days. A pure, viable capillary explant was obtained by microdissection and gentle agitation; the more rapidly occurring postmortem changes in the surrounding nervous tissue of the retina facilitated separation of the vascular explants. Factor VIII indirect immunofluorescent staining revealed that 85% to 90% of the cells harvested from capillaries of 3- to 5-day-old cadaver eyes and all cells cultured from 1-week-old postmortem eyes reacted positively, indicating their endothelial nature. High-glucose medium caused degenerative changes in the cells of the initial explant as well as in the cells of confluent cultures within 24 to 72 hours. The cytotoxic effect of glucose was manifested by accumulation of PAS-positive granules, cytoplasmic vacuolation, and eventual cell degeneration.     

Phospholipids and acylglycerols biosynthesis and 14CO2 production from [14C]glycerol in the bovine retina: the effects of incubation time, oxygen and glucose

When the entire bovine retina was incubated in 95 μm [U-¹⁴C]glycerol, the precursor penetrated the tissue and it was used for the de novo biosynthesis of glycerolipids. A minor proportion of the labeled glycerol turned to ¹⁴CO₂. The following labeling sequence was suggested: glycerol phosphorylation, double acylation, phosphatidate, diacylglycerol and triacylglycerol. Under aerobic conditions, a remarkable channelling of the label towards triacylglycerol biosynthesis was found. Comparatively, much lower specific activities were encountered in all the phosphoglycerides, phosphatidylinositol biosynthesis attaining the highest rate. At an early incubation time, the peak labeling of total phosphoglycerides was closely followed by phosphatidate radioactivity. The phosphoglyceride content slightly decreased in retinas incubated in the absence of added glucose. Anoxia, however, resulted in a diminution of the concentration of the diacylglycerophosphatides of choline and of ethanolamine and in a significant increase of their monoacyl derivatives. In an incubation medium devoid of glucose, the specific activity in the diacyl and monoacylglycerophosphatides of choline and ethanolamine was significantly raised. Furthermore, there was a dramatic decrease in the triacylglycerol biosynthesis without modification of glycerol incorporation in the polar lipids and augmented the labeling in the lipid-free tissue residue. Anoxia abolishes the uptake of glycerol in retina lipids.