Detection of virulence factors in Serratia strains isolated from contact lens‐associated corneal ulcers

Purpose: This paper reports on the microbiological findings pertaining to three Serratia isolates from soft contact lens‐related corneal ulcers, which represent a complication of contact lens wear reported with increasing frequency. Methods: Bacterial identification and antibiotic susceptibility testing were performed using the Vitek system. Serratia’s ability to form biofilm, produce gelatinase, elastase and alkaline protease, and invade human corneal epithelial (HCE) cells was investigated. The isolates’ susceptibility to the following disinfectants was tested: (a) ReNu MultiPlus^®, containing polyaminopropyl biguanide 0.0001%; (b) Opti‐Free Express^®, containing polyquaternium‐1 0.001% and myristamidopropyldimethylamine 0.0005%; (c) Opti‐Free Replenish^®, containing polyquaternium‐1 0.001% and myristamidopropyldimethylamine 0.0005%, and (d) Oxysept Comfort^®, a one‐step 3% hydrogen peroxide‐catalase system. Results: Two Serratia marcescens and one Serratia liquefaciens were identified. All the strains were susceptible to aminoglycosides and fluoroquinolones. No isolate formed biofilm or significantly invaded HCE cells; all produced alkaline protease and gelatinase, but not elastase. Opti‐Free Express^® and Opti‐Free Replenish^® were active against S. liquefaciens, but failed to kill the S. marcescens isolates within the minimum recommended time (6 hours). ReNu MultiPlus^® needed 6 hours to kill one strain of S. marcescens, which is 2 hours more than recommended. Conversely, Oxysept Comfort^® was always effective within the minimum recommended time (6 hours). Conclusions: The ability to produce alkaline protease and gelatinase may play a major role in the pathogenesis of contact lens‐related Serratia keratitis. Several types of contact lens solutions may be ineffective in eradicating Serratia from contaminated contact lens cases within the minimum recommended time. Only exposure to Oxysept Comfort^® was always effective against Serratia in this study.     

Correlation of 55 samples of contact lens fluid and direct swabbings of the eye surface of symptomatic wearers of contact lenses

Background. The microbiological examination of contact lens storage solutions may reveal the etiological organisms resposible for keratitis and additionally harbour environmental contaminants. Patients and methods. From 1995 to 1998, conjunctival and corneal smears and scrapes, as well as the storage solutions of 55 soft contact lens wearers were examined microbiologically in a prospective non-randomised cohort study involving 53× unilateral and 2× bilateral cases of keratitis. Results. In 42 of the in-use contact lens storage solutions, the following organisms were found: 8×Bacillus sp., 7×P. aeruginosa and 8× other pseudomonads, 6×Alcaligenes xylosans, 4×Serratia liquefaciens, 4×Enterobacter sp., 4× coagulase negative staphylococci, 3×Serratia marcescens, 3×S. aureus, 2×Streptococcus faecalis, 1×Klebsiella oxytoca and 5×Acanthameba. A significant correlation of ocular cultures was found for Pseudomonas, Enterobacter, Serratia, S. aureus, S. faecalis and Acanthameba, but not for Bacillus, Alcaligenes, and coagulase negative staphylococci. Fungi were not found. The more pronounced the clinical picture, the more Gram-negative bacteria were isolated. Conclusions. The microbiological examination of soft contact lens storage solutions of 55 keratitis patients confirmed that the microbes responsible were to be found in the fluid, in addition to environmental contaminants.     

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.     

Adhesion of Pseudomonas aeruginosa ocular isolates to mucin

The purpose of this study was to compare the adhesion of Pseudomonas aeruginosa ocular isolates to mucin. An adhesion assay was developed using biotin‐labelled P. aeruginosa strains (two corneal ulcer, two acute red eye, one asymptomatic and one standard strains) incubated with porcine gastric mucin immobilized on a nitrocellulose membrane. The adhesion was semiquantified using densitometry. The results showed that all P. aeruginosa strains tested were able to adhere to mucin to various extents with three strains (one corneal ulcer, one acute red eye, one asymptomatic) binding significantly greater than the negative control (P < 0.1). Results suggest that ocular strains of P. aeruginosa strains differ in their adhesion to mucin but this did not correlate with the pathogenic origin of the strain. It is concluded that the adhesion of P. aeruginosa strains to mucin alone may not be a principal determinant of pathogenesis but may be a contributing factor along with other bacterial virulence traits.     

Inhibitory effects of ovomacroglobulin on bacterial keratitis in rabbits

We studied the inhibitory effects of chicken egg-white ovomacroglobulin (ovoM) on keratitis induced by 56000-Da protease (56 KP) of Serratia marcescens and by elastase (PE) and alkaline protease (PAP) of Pseudomonas aeruginosa. The effects of ovoM on the serratial and pseudomonal keratitis in rabbits were also elucidated. In one model, four drops of 56 KP, PE, or PAP (1 mg/ml) were applied to wounded corneas of eight eyes. Thereafter, 80 l ovoM (10 mg/ml) was dropped into four eyes and 0.01 M phosphate-buffed 0.15 M saline (pH 7.4) into the other eyes as a control. The other in vivo test system involved intrastromal injection of S. marcescens or P. aeruginosa, by which each sample (10⁵–10⁷ colony-forming units) mixed with ovoM was injected into one cornea and the other cornea received organisms without ovoM. OvoM completely inhibited the activity of these bacterial proteases in vitro and reduced corneal destruction in experimental keratitis in rabbits. In addition, greatly accelerated wound healing was observed.Part of this work was presented at the 1989 meeting of the Association for Research in Vision and Ophthalmology in Sarasota, Florida Offprint requests to: H. Maeda     

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.     

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.     

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.     

Endogenous Serratia marcescens Endophthalmitis with Dark Hypopyon: A Case Report and Review

A case of endogenous Serratia marcescens endophthalmitis in a patient with diabetes, end-stage renal disease, and an indwelling venous catheter is reported. The patient presented with a tan hypopyon and elevated intraocular pressure. Diagnosis was established by positive blood, vitreous, conjunctival, and catheter tip cultures. After a deteriorating course the eye was enucleated. Gross and histopathologic examination revealed the presence of a dark hypopyon with iris necrosis and pigment dispersion and possible spontaneous globe perforation. This is the eleventh reported case of endogenous Serratia endophthalmitis. Previous association of a pink hypopyon and of pigmented vitreous fluid and Serratia endophthalmitis has been reported. This is the first case of dark hypopyon in endogenous Serratia marcescens endophthalmitis reported in the medical literature. Previous entities associated with dark hypopyon have been limited to intraocular melanoma and Listeria monocytogenes endophthalmitis. Dark hypopyon in the appropriate clinical setting may be useful in aiding diagnostic and therapeutic decisions.     

Antimicrobial peptides: a potential role in ocular therapy

Bacterial pathogens are often involved in contact lens‐related adverse responses. This study aimed to find antimicrobial peptides and proteins that effectively eradicate or inhibit ocular bacteria. The antimicrobials were screened against Gram‐ negative and Gram‐positive bacteria originating from ocular sources. The viability of these ocular bacteria was measured after exposure to the peptides and proteins. Two conditions were used to grow bacteria, low nutrient phosphate‐buffered saline and high nutrient tryptone soya broth. Samples were taken at different times up to 48 h. In low nutrient conditions, protamine was found to be the most effective against all strains. Melittin was very effective against all strains except Serratia and one Pseudomonas isolate which were partially affected. In high nutrient condition, only melittin was effective in killing Staphylococcus aureus. Protamine and the combination of protamine and melittin had the greatest effect in eradicating the bacteria tested in low nutrient condition. Protamine alone and its combination with melittin may have potential therapeutic agents for ocular infections in an era of emerging antibiotic resistance.     

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.     

Comparison on conjunctival sac bacterial flora of the seniors with dry eye in Ganzi autonomous prefecture

AIM: To compare the bacterial flora in palpebral conjunctiva of xerophthalmia seniors of Tibetan, Yi and Han, and analyze the differences and similarities of the bacteria.METHODS:The test subjects were selected from 2 Tibetan, 2 Yi and 3 Han populated places, respectively. Total 222 seniors (444 eyes) with dry eye were examined. Secretion was collected from the palpebral conjunctiva of the subjects and then inoculated onto a blood agar plate. After 48h of incubation, the bacteria were examined for the differences and similarities between different ethnics.RESULTS: There was no significant difference (P>0.05) of Gram stain characterization, dominant bacteria and number of the bacterial species present in oxrophthalmia patients among Tibetan, Yi and Han nationalities. The bacteria presented in all groups include staphylococcus epidermidis, corynebacterium, micrococcus luteu, intracellular bacteria sphingomonas, pseudomonas aeruginosa. The bacteria detected from the two of three ethnic groups were staphylococcus aureus, staphylococcus haemolyticus, escherichia coli, kytococcus sedentarius, streptococcus angina, micrococcus lylae, and staphylococcus heads. The incidence rate of bacteria-associated dry eye in Tibetan population was significantly lower than that of Han and Yi population.CONCLUSION:There is no significant difference in the bacteria flora of palpebral conjunctiva observed among dry eye elder populations of Tibetan, Yi and Han people. All of staphylococcus epidermidis, corynebacterium, micrococcus luteu, intracellular bacteria sphingomonas, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus haemolyticus, escherichia coli, kytococcus sedentarius, streptococcus angina, micrococcus lylae and staphylococcus heads are common bacteria flora of the three nationalities inhibiting in this area.     

Corneal Ring Infiltrates Caused by Serratia marcescens in a Patient with Human Immunodeficiency Virus

PurposeTo describe corneal ring infiltrates caused by Serratia marcescens in a patient with human immunodeficiency virus (HIV-1) who wore contact lenses.MethodsA case study of a patient with keratitis due to an infection caused by S. marcescens and exhibiting corneal ring infiltrates was reviewed for history, clinical manifestation, microscopic study, and management.ResultsA 29-year-old man who had a history of contact lens wear and HIV-1 infection was admitted to hospital because of blurred vision, redness, and corneal infiltrates in the shape of a ring in the left eye. The visual acuity (VA) in both eyes was hand movement (uncorrected). Corneal scrapings were performed. The culture results of the corneal specimens revealed S. marcescens. The culture results of the contact lens disclosed the same organism. The corneal ulcer responded well to treatment with topical gentamycin sulfate 14 mg/ml. The final VA remained hand movement.ConclusionsS. marcescens can cause ring infiltrates in a HIV-1 patient who wears contact lenses. The treatment result for S. marcescens keratitis in a HIV-1 patient who wore contact lenses was favorable after intensive use of fortified topical antibiotics.Key Words: Serratia marcescens, Corneal ring infiltrates, Human immunodeficiency virus, Contact lens     

The pathogenesis of bacterial keratitis: studies with Pseudomonas aeruginosa

Bacterial keratitis is a sight‐threatening corneal disease that is most commonly associated with the extended wear of soft contact lenses. Over the past decade, we have investigated the pathogenesis of infectious keratitis involving the opportunistic pathogen Pseudomonas aeruginosa. Our research has focused on understanding the respective roles of bacteria and host in the establishment of this infection. Here, we provide a current perspective on P. aeruginosa keratitis, reviewing some of the research developments that have helped shape our views on the mechanisms by which pathogen and host response cause corneal disease. P. aeruginosa may provide a model for the pathogenesis of bacterial keratitis and help further elucidate the complex array of host factors that normally protect the cornea from infectious agents.     

Epidemiological characteristics of microbiological results on patients with infectious corneal ulcers: a 13-year survey in Paraguay

Background This is a retrospective, chart-reviewed study of patients diagnosed with infectious corneal ulcers at the Ophthalmology Department of the National University of Asunción in Paraguay. The microbiological culture results are described, as well risk factors for the development of fungal keratitis.Methods After obtaining approval from the Institutional Review Board, an analysis of medical charts from 1988 to 2001 was conducted and 660 patients were identified to have been diagnosed with infectious corneal ulcers due to bacteria or fungi. Demographic data were recorded, including age, gender, occupation and geographic location of their home and work (city or rural). Other information collected included the history of the presenting illness, past and current use of ocular medications and whether or not they had a history of trauma or contact lens use. Each patient had an eye examination performed by an ophthalmologist and corneal scrapings were obtained for cultures in all cases. Microbiologic culture results were analyzed.Results Twenty-one percent (136/660) of the specimens collected from the patients conjunctiva and cornea were sterile in all culture media. Of the 524 (79%) positive cultures, 267 were due to bacteria (51%), 136 to fungi (26%), and 121 (23%) cultures yielded both fungi and bacteria. Of the 430 isolated bacteria approximately 25% (103) were coagulase negative Staphylococcus, followed by 23% (94) Staphylococcus aureus, 14% (60) Pseudomonas aeruginosa and 13% (56) Streptococcus pneumoniae. Acremonium species accounted for 40% (79) of all fungi identified, followed by Fusarium species (15%) (41). Approximately two-thirds of the patients were male (n = 435). For those patients for whom a history was available, approximately half had a history of trauma. Of these, half of these again involved foreign bodies. Over-the-counter medications were used commonly, and most of those patients had a delay in diagnosis of over 1 week. Risk factors for fungal keratitis as opposed to bacterial keratitis were male gender, agricultural occupation, age between 30 and 59, history of trauma and self-medication.Conclusions The results of this study provide demographic data on patients with infectious corneal ulcers in Paraguay. Common causes of such ulcers are both bacteria and fungi. Most patients had self-medicated, and most had delayed seeking professional medical care.This article was presented in part at the Paraguayan Congress of Infectology, Asunción, Paraguay, in 0ctober 2001.     

Regulatory role of IL-1β in the expression of IL-6 and IL-8 in human corneal epithelial cells during Pseudomonas aeruginosa colonization

Pseudomonas aeruginosa is a virulent pathogen and is frequently associated with bacterial keratitis. Recent studies have shown that high levels of interleukin (IL)‐1β and macrophage inflammatory protein‐2 are associated with the severity of corneal infection. Interleukin‐1β is a principal inflammatory mediator. Understanding the regulatory role of IL‐1β would provide better understanding of host responses during P. aeruginosa corneal infection. A human corneal epithelial (HCE) cell line and three P. aeruginosa strains were used in this experiment. Confluent HCE cells were challenged with P. aeruginosa and monoclonal antihuman IL‐1β antibody (IL‐1β mAb). The culture supernatants were collected for measuring cytotoxicity and protein levels of IL‐1β, IL‐8 and IL‐6 by enzyme‐linked immunosorbent assay. Results showed that HCE cells expressed low levels of IL‐1β and high levels of IL‐6 and IL‐8 during P. aeruginosa colonization. Paer1‐colonized HCE cells produced higher levels of IL‐1β, IL‐6 and IL‐8 protein compared to those produced by 6206‐ and 6294‐ colonized HCE cells. Administration of IL‐1β mAb decreased the production of IL‐8 and IL‐6. In conclusion, P. aeruginosa‐colonized HCE cells produced low levels of IL‐1β and high levels of IL‐6 and IL‐8. Neutralizing IL‐1β protein significantly downregulated the production of IL‐8 and IL‐6.     

Adhesion and growth of Serratia marcescens on artificial closed eye tears soaked hydrogel contact lenses

The adhesion to hydrogel contact lenses and growth of Serratia marcescens on artificial tear fluid (ATF) soaked lenses was investigated. Results indicated that a corneal ulcer isolate adhered more avidly to lenses; ATF increased adhesion for all strains tested. The contact lens induced acute red eye (CLARE) isolate adhered poorly; however; it grew to a larger extent on ATF-coated lenses. The ability of the corneal ulcer isolate to adhere to lenses may be an important factor in its pathogenicity whereas the ability of the CLARE isolate to grow on the lens in the presence of antimicrobial tear proteins may be important in the development of inflammation.     

Secretory immunoglobulin A in tears: functions and changes during contact lens wear

Introduction : Secretory immunoglobulin A (sIgA) is the predominant immunoglobulin in tears. The role of sIgA in defending the eye against pathogens has not been established clearly. There have been conflicting reports about the effect of contact lens wear on the concentration of sIgA in tears. This study was conducted to elucidate the role of sIgA in ocular defence and to determine the effect of contact lens wear on sIgA concentration. Methods : Tears were collected from contact lens wearers and non-wearers using microcapillary tubes. The concentration of sIgA was evaluated using an in-house ELISA. The specificity of sIgA to a strain of P. aeruginosa was examined using a fluorescent assay and the ability of neutrophils to phagocytose sIgA coated bacteria were assessed by plate counts. Results : Tears contained sIgA that reacted to P. aeruginosa. P. aeruginosa coated with sIgA was phagocytosed by the neutrophils. The level of sIgA and the level of sIgA specific to P. aeruginosa in the tears of contact lens wearers were significantly reduced. Conclusions : These results indicate that contact lens wear significantly alters the level of sIgA in tears which may lead to changes in the ability of the ocular surface to defend itself against potential pathogens.     

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。