Production of N-acyl homoserine lactones by Gram-negative bacteria isolated from contact lens wearers

The purpose of this study was to investigate the production of N‐acyl‐homoserine lactone (AHL) signal molecules in ocular Gram‐negative bacteria. A total of 91 ocular strains isolated from contact lens adverse response patients and asymptomatic subjects were used in the study. These included Acinetobacter, Aeromonas hydrophila, Escherichia coli, Haemophilus influenzae, Klebsiella oxytoca, Pseudomonas aeruginosa, Serratia liquefaciens, Serratia marcescens, and Stenotrophomonas maltophilia. The biosensor strains Chromobacterium violaceum mutant CV026 and Agrobacterium tumefaciens A136 were used for detection of AHL signal molecules. The majority of A. hydrophila, P. aeruginosa, and S. liquefaciens strains produced more than one AHL molecule. Serratia marcescens strains were AHL positive only under detection of A136. The rest of the test species did not show any AHL production under the current detection system. These findings indicate that AHL‐ mediated quorum‐sensing systems are present in some of the ocular bacteria, and the different signal molecules may be involved with the quorum‐sensing pathway in the other bacterial species.     

Detection and specificity of anti-Staphy/ococcus intermedius secretory IgA in human tears

Purpose: Secretory IgA (slgA) is the predominant immunoglobulin present in tears that protects the ocular surface against various antigens. Staphylococcus intermedius is a member of the normal ocular microbiota. Methods: The presence and the specificity of immunoglobulin A to S. intermedius was determined by fluorescent assay, ELISA and western blots. Results: Three immunodominant antigens of S. intermedius were detected of 145, 127 and 61 kDa. Staphylococcus inter-med/us-specific IgA cross reacted with Staphylococcus aureus but not with Gram negative bacteria. Conclusions: This indicates that specific IgA may play an important role in the protection of the eye by limiting the levels of Gram positive normal microbiota and defending against the more pathogenic S. aureus.     

Microbial keratitis in Waikato, New Zealand

Background: Bacterial keratitis is a potentially sight‐threatening condition. This study is performed to identify the common causative organisms for bacterial keratitis in Waikato region and the antibiotic sensitivities to these organisms. Design: Retrospective, observational, case series. Participants: The microbiology records of all patients with bacterial keratitis who presented to the Ophthalmology department, Waikato Hospital, New Zealand between January 2003 and December 2007. Methods: The corneal scrape results were reviewed. Antibiotic sensitivity for the organism was tested following National Committee for Clinical Laboratory Standards (NCCLS) method. Main Outcome Measures: In vitro laboratory susceptibility testing of ocular isolates to various antibiotics. Results: A total of 265 scrapes were performed. Gram stain was positive in 35 (13.2%) eyes. Positive culture was seen in 174 (65.6%) scrapes; 78.2% were Gram‐positive and 20.2% were Gram‐negative organisms. Most common Gram‐positive organisms were coagulase‐negative Staphylococci (40.8%) and Staphylococcus aureus (11.5%). Most common Gram‐negative organisms were Moraxalla species (8.0%) and Pseudomonas aeroginosa (3.4%). Of the bacterial organisms 99% were sensitive to ciprofloxacin. All Gram‐negative organisms and 95.5% Gram‐positive organisms were sensitive to tobramycin; 96.6% Gram‐positive organisms and 98.3% Gram‐negative organisms were sensitive to cefuroxime. Conclusions: Our results are comparable to other regions in New Zealand but the incidence of coagulase‐negative Staphylococcus is much higher in this region compared with other New Zealand studies. It seems appropriate to start patients with corneal ulcers initially on fluoroquinolone monotherapy while awaiting culture results.     

Interactions between the constitutive host defences of tears and Staphylococcus epidermidis

As part of the normal microbiota of the external eye, Staphylococcus epidermidis has probably developed strategies to overcome tear defences. The interaction of this bacteria with constitutive tear proteins was examined. Isolates of S. epidermidis grown in 10% serum or artificial tear fluid containing lysozyme or lactoferrin were resistant to the action of these proteins. Using ELISA, cell wall binding of C3, vitronectin and lactoferrin differed quantitatively between strains and in closed-eye compared to open-eye conditions. No differences were observed between ocular and non-ocular strains. This suggests that ocular isolates originate from the general host microbiota and S. epidermidis isolates are resistant to individual constitutive tear proteins.     

Growth of Gram-negative bacteria in a simulated ocular environment

Purpose: Gram-negative bacteria are major pathogens in the ocular environment, especially when contact lenses are worn. The source of many of these organisms, including Pseudomonas aeruginosa, is thought to be environmental. Methods: A defined medium is described here that resembles both tear fluid and the environment they are derived from. Results: Gram-negative ocular bacteria were found to grow successfully in this medium. Bactericidal tear proteins were added to this medium and pathogenic strains were not only able to resist their effects but were able to use them for growth. Conclusion: Utilizaton of tear proteins for growth may be an important virulence factor of pathogenic bacteria in the ocular environment.     

新生儿眼部感染情况分析

目的:研究2014/2018年浙江苍南县新生儿眼部感染病原菌分布、常用抗生素耐药性及流行特征,为临床病情的预防、诊断与治疗提供依据。方法:将本院2014-01/2018-12新生儿门诊294份眼部标本中分离所得108株病原菌进行鉴定、常用药物敏感试验和流行病特征汇总分析。结果:眼部分泌物标本294份中病原菌检出率为36.7%(108/294)。主要菌群包括表皮葡萄球菌48株(44.4%),金黄色葡萄球菌16株(14.8%),大肠埃希菌24株(22.2%),淋病奈瑟菌12株(11.1%),类白喉棒状杆菌5株(4.6%),肠球菌属2株(1.9%),链球菌属1株(0.9%)。主要病原菌对阿米卡星和奈替米星具有高度敏感性,对洁霉素和庆大霉素活性较差。对喹诺酮类抗生素活性较好。结论:新生儿眼部感染的致病菌以葡萄球菌属为主,临床常用眼部抗菌药物对各类病原菌具有良好的抗菌活性。     

Human antimicrobial peptides in ocular surface defense

Sight depends on the passage of light through the transparent cornea and being focused on the fovea. Its exposed position renders it vulnerable to microbial infection. The cornea has developed a wide array of defense mechanisms against infection, of which endogenous antimicrobial peptides (AMPs) are key. AMPs are essentially small molecular weight cationic peptides with a wide range of activity against virus, bacteria, fungi and parasites. Some proteins such as RNases and S100As are also included in this group. Several AMPs act synergistically allowing low expression of multiple AMPs to act efficiently. AMPs also have a range of non-microbicidal functions and serve as signaling molecules, immunomodulators; show anti-tumour activity, and influence vascularization and wound healing. Different toll-like receptors (TLR) have been implicated in the preferential induction of specific AMPs. A range of bacteria, including mycobacteria tuberculosis, viruses including herpes virus, fungi and parasites including acanthamoeba, that cause ocular infections have been shown to induce specific AMPs via TLR activation. Non-TLR mediated induction of AMP expression can occur and several molecules such as L-isoleucine, sodium butyrate, vitamin D3, phenylbutyrate, vasoactive intestinal peptide, and etinostat have been identified in this regard. Given the rising microbe resistance to antibiotics, the slow rate of development of new antibiotics and the limited access to effective antibiotics by patients living in the developing world, an ideal solution would be to find AMPs that are effective singly or in combination with each other or other antimicrobial proteins to reduce, if possible eliminate reliance on antibiotics alone.     

Overview of mechanisms of antibiotic resistance in Pseudomonas aeruginosa: an ocular perspective

Treatment of Pseudomonas aeruginosa eye infections often becomes a challenge due to the ability of this bacterium to be resistant to antibiotics via intrinsic and acquired mechanisms. Transfer of resistance due to interchangeable genetic elements is an important mechanism for the rapid transfer of antibiotic resistance in this pathogen. As a result, drug‐resistant strains are becoming increasingly prevalent worldwide. This review systematically analyses data from recent publications to describe the global prevalence and antibiotic sensitivity of ocular P. aeruginosa. Thirty‐seven studies were selected for review from PubMed‐based searches using the criteria ‘microbial keratitis OR eye infection AND Pseudomonas aeruginosa AND antibiotic resistance’ and limiting to papers from 2011 onward, to demonstrate the antibiotic resistance from isolates from around the world. Subsequently, we reviewed the ways in which P. aeruginosa can become resistant to antibiotics. Both the rate of isolation of bacteria in general (79 per cent of cases), and prevalence of P. aeruginosa (68 per cent of all isolates) were highest in contact lens‐related microbial keratitis. The average resistance rate to common ocular antibiotics such as ciprofloxacin (9 per cent), gentamicin (22 per cent) and ceftazidime (13 per cent) remained relatively low. However, there were large variations in resistance rates reported in studies from different countries, for example resistance to ciprofloxacin reached up to 33 per cent. We next reviewed the types of mobile genetic elements (MGEs) such as plasmids, integrons and transposons that are frequently associated with drug resistance in P. aeruginosa. MGEs are important for the transmission of resistance to beta‐lactams and aminoglycosides and recently have been shown to be potential factors for the transmission of fluoroquinolone resistance. Studies on the molecular mechanisms of resistance transfer in ocular P. aeruginosa have begun to be reported and will provide valuable information on the emergence of new antibiotic resistance and potential to treat resistant strains.     

Postoperative ocular infections: an analysis of laboratory data on 750 cases

From 1981-1982, 750 clinical specimens from patients who developed postoperative infections were processed in the ocular microbiology laboratory. Bacterial cultures were positive in 71.0%, fungal in 1.3% and another 1.4% yielded both. The remaining 26.3% were sterile. Infections were due chiefly to Gram positive organisms (63.6%); only 6.4% were due to Gram negative bacteria while 0.9% were due to both. Staphylococcus aureus (52.0%) was the most common isolate, followed by S epidermidis (37.2%). Pseudomonas aeruginosa and Acinetobacter calcoaceticus were the next frequent pathogens. Aspergillus was the most common fungus among the fungal isolates. The highest number of infections followed cataract extraction and keratoplasty. A vast majority of postoperative infections seem to be occurring with hospital acquired strains. Cloxacillin seems to be the most effective remedy for treating staphylococcal infections and polymyxin B for infections due to Pseudomonas. Chloramphenicol appears to be as effective as gentamicin for treating all infections.     

Some potential pathogenic traits of gram-negative bacteria isolated during ocular inflammation and infections

Background : Bacterial colonisation of contact lenses is believed to be important in the production of microbial keratitis and acute inflammatory reactions. The aim of the current study was to examine strains isolated from ocular infections and non-infectious ocular inflammatory conditions for their ability to adhere to contact lenses and epithelial cells and to stimulate the release of chemotactic substances from epithelial cells. Methods : Bacterial adhesion to contact lenses and bovine corneal or conjunctival cells was studied by adhesion assay. Agglutination of human red blood cells by bacteria was demonstrated by haemagglutination. The chemoattractants released from corneal epithelial cells exposed to bacteria was assessed by polymorphonuclear leukocytes (PMN) chemotaxis. Results : Strains of P. aeruginosa adhered better on the contact lenses than strains of other gram-negative bacteria (p = 0.004). H. influenzae strains isolated from conjunctivitis produced haemagglutination. Leukotriene B₄ was released from corneal epithelial cells after stimulation by the gram-negative bacteria. Conclusion : This study has identified several potential pathogenetic traits of gram-negative bacteria that may contribute to ocular infection and/or inflammation. It has been estimated that 70 per cent of microbial keratitis cases associated with contact lens wear involve P. aeruginosa and this study has shown that this bacterial species adheres in large numbers to contact lenses. This increased adhesion to contact lenses may be one of the reasons for the preponderance of this species in these infections. The finding that strains could stimulate corneal epithelial cells to release chemotactic factors that specifically recruit PMN indicates a mechanism for producing corneal infiltration.     

Suppurative keratitis in rural Bangladesh: the value of Gram stain in planning management

External eye diseases which result in corneal scarring are an important cause of blindness in Bangladesh and at the Chittagong Eye Infirmary and Training Complex (EITC) over 200 cases of suppurative keratitis are managed each year. We reviewed the records of 127 cases of microbial keratitis to determine the relative contributions of Gram stain and culture to diagnosis of the causative organism. There were 107 culture-proven cases of microbial keratitis amongst the 127 patients in this study. Gram stain was positive in 89 cases which represents 70% of the total and 83% of all culture-proven cases. Streptococcus pneumoniae and Pseudomonas sp were the commonest bacteria isolated and Aspergillus sp and Fusarium sp the commonest fungi. In 20 cases (16%) no organism was isolated on Gram stain or culture. Our results support the use of both Gram stain and culture in isolation of the causative organism in cases of suppurative keratitis in Bangladesh. However the low cost of Gram stain and its useful recovery rates for both bacteria and fungi support its use as an initial investigation for microbial keratitis at the secondary level of eye care in rural Bangladesh.     

Serotype and adhesion of Pseudomonas aeruginosa isolated from contact lens wearers

The purpose of the present study was to correlate the serotypes of Pseudomonas aeruginosa to the bacterial adhesion to contact lenses and human corneal epithelial cells. Twenty‐three strains isolated from contact lens wearers were used for the study. The bacterial serotypes were examined with a P. aeruginosa antisera kit. The attachment of bacteria on contact lenses or human corneal epithelial cells was determined by counting the number of adhered bacteria after incubation of the bacteria with contact lenses or corneal epithelial cells. The 23 ocular isolates belonged to seven serotypes. Strains of serotypes I, G and E were the three dominant serogroups and were more adhesive to contact lenses compared with other groups of the bacteria. The bacterial serotypes and the clinical sequelae were not strongly related. These results indicate that the surface characteristics of bacterial serotypes are related to the bacterial adhesion to the surface, but the pathogenesis of the bacteria may result from multiple factors.     

新型OxyPlateTM厌氧系统隔离眼部厌氧菌的评估

目的:厌氧细菌可引起眼部感染,我们测试OxyPlate^TM厌氧系统(OxyPlate^TM Anaerobic System,OXY)隔离可引起眼部疾病的厌氧细菌。方法:OXY不需要直接的厌氧条件（比如厌氧袋,罐）,将其与常规的厌氧袋培养基相比。琼脂培养基上眼部厌氧细菌菌株在好氧和厌氧条件下（厌氧袋）行标准的菌落计数:（1）OXY（好氧）;（2）5%羊血（sheep blood,SB）;（3）巧克力琼脂;（4）Schaedler琼脂。测试的眼部体外培养细菌来自眼内炎,泪囊炎,包括10个丙酸杆菌和3个放线菌种类。在每个培养条件下,每个细菌菌落计数隔离,排名从大到小,并在非参数比较下确定最佳的培养条件。结果:所有的厌氧条件对于厌氧菌株呈阳性反应。厌氧菌在有氧条件下的SB和Schaedler的琼脂中无法增长。痤疮丙酸杆菌在巧克力琼脂中生长稀疏。作为一种厌氧系统,在厌氧袋SB分离比OXY（P=0.0028）和巧克力琼脂（P=0.0028）分离出更多的菌落数。结论:虽然OXY经测试并没比其他的厌氧系统更高效,它似乎是一个合理隔离厌氧细菌的替代方法。其琼脂培养基在一个专门设计的盘并不需要厌氧袋使得OXY优于其他厌氧系统。     

The pharmacokinetics of linezolid in the non-inflamed human eye

AIM: This study describes the ocular pharmacokinetics of linezolid, an antibiotic with broad spectrum activity against those Gram positive bacteria that are the most frequent cause of postoperative endophthalmitis. METHOD: Patients undergoing routine cataract surgery were given a single oral 600 mg dose of linezolid at a variable time before surgery. Aqueous and serum levels of linezolid were assayed by high performance liquid chromatography, and a pharmacokinetic curve constructed from the pooled results. RESULTS: Orally administered linezolid rapidly achieves levels in the aqueous of non-inflamed eyes that exceed the concentration required to kill Gram positive bacteria (maximum mean concentration 6.8 (SD 1.2) microg/ml at 2-4 hours post-dose). An effective concentration is maintained for at least 12 hours, the standard interdose interval for this antimicrobial. CONCLUSION: Linezolid offers the possibility of a rapid, oral approach to effective treatment of most cases of postoperative endophthalmitis, with the potential of improving visual outcome.     

Bacteriological analysis in the management of conjunctivitis. Comparison of antibiotic resistance between 1982 and 2008

ObjectiveTo demonstrate the need for bacteriological analysis for the rational treatment of infections of the corneal surface, including conjunctivitis.MethodsThe results of 1,970 consecutive bacteriological analysis obtained from 2001 to 2008 in patients with ocular hyperemia and discharge were analysed and compared with a similar study done in 1982 in the same geographical area.ResultsBacterial growth was obtained in 1,044 cases (53%). The most frequent bacteria were Staphylococci spp. (56.6%), followed by Streptococci spp. (21.4%), Haemophili spp. (12.1%), and other gram-negative bacteria (9.9%). No antibiotic was effective against all the bacteria isolated. The frequency of resistant bacteria against neomycin, tobramycin, erythromycin and gentamicin was significantly increased with respect to the study of 1982.ConclusionsBacteriological analysis of conjunctival specimens are necessary to ensure the choice of an effective antibiotic against bacteria of the ocular surface when the initial treatment fails.     

Surfactant Protein D in Pseudomonas aeruginosa Keratitis

Purpose: To determine the importance of surfactant protein D in Pseudomonas keratitis. Methods: The surfactant D status of wild-type and surfactant D-deficient Black Swiss mice was confirmed by PCR reactions and immunoblot assay. Mouse corneas were infected with one of three strains of P. aeruginosa. At 1, 2, 3, and 6 days postinfection, eyes were scored by slit-lamp examination and bacteria per cornea quantified. Results: Infected wild-type mice had slit-lamp scores on 3 and 6 days postinfection that were significantly lower than those of surfactant D-deficient mice (p ≤ .0185) and only wild-type mice recovered from the infection. On day 6 postinfection, the number of bacteria per cornea was found to be significantly lower in wild-type mice as compared to surfactant D-deficient mice (p ≤ .0233). Conclusions: Surfactant D is important to the ocular innate host defense against Pseudomonas keratitis.     

Profile of sight‐threatening infectious keratitis: a prospective study

Purpose: To prospectively study patients presenting with sight‐threatening corneal ulcers with a view to identify the predisposing factors, causative organisms, clinical signs and treatment outcomes. Methods: Prospectively, over 3‐year period, all cases with serious infective keratitis presenting to Queens Medical Hospital in Nottingham, UK, were recruited. Detailed information on the aetiology, culture results, signs & symptoms, the treatment given and the patient’s response was collected and statistically analysed. Results: One hundred and forty‐three eyes of 129 patients were enrolled. Thirty‐one patients were managed as out‐patients, and 98 were treated as in‐patients. The mean duration of admission was 9 ± 13 days but was significantly higher in older patients and in Acanthamoeba keratitis cases. The important risk factors were ocular surface disease (32%), contact lens wear (26%) and previous ocular surgery (20%). Old age, deep infiltration, steroid use and poor initial vision were risk factors for prolonged course of treatment in bacterial keratitis. Corneal scrapings were done in 89% of the cases, but positive results were obtained only in 41.7%. Staphylococcus aureus was the most common isolated bacteria (18.8%). Acanthamoeba and Pseudomonas aeruginosa were the second and third common causative organisms (16.6% and 15%, respectively). Overall, 8.3% needed corneal grafting, which survived in 83.3% and eradicated infection in 100%. Conclusion: Microbial keratitis is an important cause of ocular morbidity. Previous ocular disease is an important predisposing factor. Old age, steroid use and poor presenting visual acuity are important prognostic indicators. Corneal grafting is an effective option for managing recalcitrant corneal infections.     

Inhibition of ocular neovascularization by a novel peptide derived from human placenta growth factor‐1

Purpose: To evaluate the effect of ZY1, a novel 21‐amino acid peptide from human placenta growth factor‐1 (PlGF‐1), against ocular neovascularization, and to study its possible toxicity to the retina and the underlying mechanism of antiangiogenic effect. Methods: MTS assays, a modified Boyden chamber and Matrigel^? were used to evaluate the effect of ZY1 on the proliferation, migration and tube formation of RF/6A rhesus macaque choroid‐retina endothelial cells induced by vascular endothelial growth factor (VEGF) in vitro. The antiangiogenic effect of ZY1 was also studied with corneal micropocket angiogenesis assays and oxygen‐induced retinopathy (OIR) assays in mice. Electrophysiological tests and histological examinations were used to study the possible toxicity of ZY1 against mouse neuroretina. Competitive ELISA and Western blotting were performed to elucidate the underlying mechanism of ZY1. Results: ZY1 inhibited VEGF‐induced RF/6A proliferation, migration and tube formation. It also inhibited ocular neovascularization when applied to the corneal micropocket angiogenesis assays and OIR assays in mice. Electrophysiological tests and histological examinations revealed no evident functional or morphologic abnormalities in mouse neuroretina after ZY1 injection. ZY1 competed for binding to VEGFR‐1 against PlGF and VEGF and inhibited VEGFR‐1/ERK/AKT activation. Conclusion: It is concluded that the novel peptide ZY1 is an effective inhibitor of ocular pathologic angiogenesis and may provide a promising alternative for ocular antiangiogenic therapy.     

Inhibition of growth factor effects in retinal pigment epithelial cells

Several agents were examined for their effect on growth factor-stimulated processes in retinal pigment epithelial (RPE) cells. DNA synthesis was assessed by 3H-thymidine incorporation in density-arrested cells using previously determined maximally effective concentrations of various growth factors with and without test substances. Cell migration was assessed in Boyden chamber assays. For each test substance, trypan blue exclusion was used to determine noncytotoxic concentrations, and the effect of several concentrations were assessed on selected growth factors. The most effective, nontoxic concentration was then used for comparisons. Two cationic proteins, protamine and histone type II B, caused inhibition of RPE chemotaxis and 3H-thymidine incorporation induced by several growth factors, but a cationic polypeptide, polylysine, did not. Protamine and histone, were particularly effective inhibitors of acidic and basic fibroblast growth factors (FGF) but not if they were exposed to cells and then removed before growth factor addition. They had no effect on serum-stimulated chemotaxis or 3H-thymidine incorporation even when used in the presence of serum. Three anionic substances, heparin, pentosan polysulfate, and suramin, also inhibited RPE chemotaxis and 3H-thymidine incorporation induced by several different growth factors. They were less effective inhibitors of the FGFs than protamine and histone but were better inhibitors of serum-induced effects. Also unlike protamine and histone, the anionic substances maintained their inhibitory effect even when removed before growth factor addition. Since migration and proliferation of RPE cells are important processes in the pathogenesis of proliferative vitreoretinopathy, these agents and their mechanism of action deserve further study for potential therapeutic applications.