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Biomedical subjects

Mark D P Willcox

Publications and source records attributed to Mark D P Willcox.

At least 19 recordsLinked to original sources
PubMed2006

The role of CXC chemokine receptor 2 in Pseudomonas aeruginosa corneal infection.

Pseudomonas is one of the leading causes of contact lens-related microbial keratitis. Despite the use of antibiotics, the host inflammatory response continues to cause damage to the cornea, which may lead to blindness. CXCR2-binding chemokines have been implicated in the pathogenesis of Pseudomonas keratitis, and the exact role of this receptor remains to be elucidated. Corneas of CXCR2 knockout and wild-type mice (Cmkar 2-/- and Cmkar 2+/+) were scratched, and 2x10(6) CFU/mL Pseudomonas 6294 or 6206 was added to corneas. Twenty-four hours postinfection, mice were killed, and eyes were harvested for enumeration of bacteria, myeloperoxidase (MPO) levels, and inflammatory mediators. Cmkar 2-/- had 20- to 100-fold more bacteria than Cmkar 2+/+ mice. There were no differences in MPO levels between gene knockout and Cmkar 2+/+ mice. Histology revealed PMN were restricted to the limbal area. Levels of CXCR2 chemokines (keratinocyte-derived chemokine and MIP-2) were elevated significantly in gene knockout mice. A lack of CXCR2 leads to an inability to control bacterial numbers as a result of the inability of PMN to reach the site of infection in the avascular cornea. These results imply that CXCR2 is critical to the extravasation of neutrophils into the avascular cornea.

Animals↗
PubMed2006

The adhesion of Pseudomonas aeruginosa to high molecular weight human tear film species corresponds to glycoproteins reactive with Sambucus nigra lectin.

Pseudomonas aeruginosa is a pathogen gaining prevalence in contact lens-related corneal ulcers. Tear outflow protects the ocular surface, where high molecular weight tear glycoproteins bind bacteria for removal from the eye. The purpose of the present study was to identify glycoproteins in human tears involved in the adhesion of ocular P. aeruginosa isolates. Basal human tears were applied to a bacterial adhesion assay involving electrophoretic separation of tear components, transfer to nitrocellulose and incubation with biotin-labelled bacteria. Glycoproteins were further characterised using lectin profiling. The results showed large-dimension agarose gels were imperative for the detection of at least four glycoproteins with a migration >200 kDa, including species not previously identified. P. aeruginosa 6294 preferentially bound to a well-defined glycoprotein near the origin of the gel that, unlike other glycoproteins >200 kDa, reacted with Sambucus nigra lectin (sialic acid alpha2-6) but not WGA lectin (N-acetylglucosamine, sialic acid alpha2-3). Adhesion did not involve free biotin label or hydrophobic interactions. Also, the pre-incubation of separated tear glycoproteins with S. nigra lectin increased subsequent adhesion of 6294 to this tear glycoprotein. The less virulent Paer1 strain showed diffuse adhesion in the S. nigra-reactive region at the gel origin. In conclusion, an overlay adhesion assay was developed that identified slow-migrating sialylated glycoprotein species in human tears preferentially bound by P. aeruginosa ocular strains, and S. nigra lectin seemed to enhance the interaction. The study provides a basis for direct investigation of bacterial adhesion to glycoproteins with an apparent migration >200 kDa in tear fluid.

Acetylglucosamine↗
PubMed2006

Type III secretion system-associated toxins, proteases, serotypes, and antibiotic resistance of Pseudomonas aeruginosa isolates associated with keratitis.

The association between possession of toxin gene-related type III secretory system, protease profiles, O serotypes, and antibiotic resistance patterns was characterized genetically and phenotypically in 46 keratitis isolates of Pseudomonas aeruginosa. There was no significant difference in exoU or exoS prevalence among the keratitis strains. Distinct protease profiles were seen in isolates harboring either exoU or exoS genes. One hundred percent (13/13) of serotype E (O:11) strains contained type III secretion system-associated cytotoxin gene exoU. Multidrug resistance was identified in 4% of Australian and 29% of Indian isolates. None of the Australian isolates was resistant to ciprofloxacin. In general, the rate of multidrug resistance in the exoU positive cytotoxic and serotype E (O:11) strains was significantly higher than in exoS positive invasive strains (p < 0.01). The results suggest that multidrug resistance may be more commonly associated with the corneal isolates of P. aeruginosa having type III secretion system-associated cytotoxin gene exoU and belonging to serotype E (O:11) group.

ADP Ribose Transferases↗
PubMed2006

Salicylic acid reduces the production of several potential virulence factors of Pseudomonas aeruginosa associated with microbial keratitis.

PURPOSE: Pseudomonas aeruginosa is a common cause of contact-lens-related microbial keratitis. This bacterium is becoming increasingly resistant to antibiotics, and even if the infection can be treated with antibiotics, damage to the cornea resulting from the combined effect of bacteria and host factors can lead to loss of vision. The purpose of this study was to test the effect of salicylic acid on the production of potential virulence factors during the growth of P. aeruginosa. METHODS: Bacterial cells were grown in a subinhibitory concentration of salicylic acid, and supernatants were collected and analyzed for presence of proteases by using zymography and hydrolysis of chromogenic substrates. The supernatants were also analyzed for the amount of acetylated homoserine lactones by using bacterial reporter strains. Pseudomonas cells from salicylic acid cultures were analyzed for their twitching and swimming motility as well as their ability to invade or cause the death of corneal epithelial cells. RESULTS: Growth in a subinhibitory concentration of salicylic acid resulted in a significant reduction in the number of bacterial cells and a reduction in the rate of the number of bacteria increasing during logarithmic growth, but the time to reach the stationary phase of growth was unchanged. These changes in growth pattern affected the amount of acylated homoserine lactones produced by P. aeruginosa 6294. Also affected by growth in salicylic acid was the ability of strain 6294 to show twitching or swimming motility. Salicylic acid also reduced the invasion of strain 6294 into corneal epithelial cells and the epithelial cell death caused by strain 6206. Furthermore, production of proteases by P. aeruginosa was significantly reduced by growth in salicylic acid. CONCLUSIONS: The results of this study clearly demonstrate that salicylic acid has a significant impact on several potential virulence factors of P. aeruginosa that may be involved in the production of microbial keratitis. These effects were probably mediated by reduction in the cell density and concomitant reduction in the quorum-sensing signaling molecules, the acylated homoserine lactones, produced by P. aeruginosa.

4-Butyrolactone↗
PubMed2006

A protective role for IL-6 in staphylococcal microbial keratitis.

PURPOSE: To determine whether interleukin-6 (IL-6) plays a protective role in Staphylococcus aureus keratitis in a gene knockout (gko) mouse model and to determine whether IL-6 may be used as a therapy to modulate host responses and control bacterial infection, thereby reducing scarring. METHODS: The eyes of IL-6 gko mice and wild-type mice were challenged topically with S. aureus and examined at 24 hours after infection. Keratitis was examined clinically and histologically. Bacterial and polymorphonuclear leukocytes (PMNs) were enumerated, and cytokine and chemokine levels were determined by ELISA. Exogenous IL-6 was administered to both IL-6 gko and wild-type mice, and clinical parameters were determined. RESULTS: IL-6 gko mice showed more severe disease, with increased bacterial counts and PMNs, than did wild-type mice. Changes in levels of chemokines and cytokines were also observed. Administration of exogenous IL-6 resulted in an improved outcome in IL-6 gko mice, with a threefold reduction in bacterial load. CONCLUSIONS: The data suggest an important regulatory role for IL-6 in modulating excessive inflammatory responses and in controlling bacterial proliferation. IL-6 may play a role in the priming and activation of neutrophils. It could represent a broad-spectrum therapy to improve outcomes in patients who have these potentially blinding infections.

Animals↗
PubMed2005

Effects of alpha-toxin-deficient Staphylococcus aureus on the production of peripheral corneal ulceration in an animal model.

PURPOSE: To examine the role of Staphylococcus aureus alpha-toxin in contact lens-induced peripheral ulceration (CLPU). MATERIALS AND METHODS: Proteolytic enzyme, hyaluronidase, alpha-toxin, and beta-toxin production by S. aureus 8325-4 and its alpha-toxin-deficient mutant (S. aureus DU1090) were examined. Using a rabbit model of CLPU, animals were fitted with hydrogel contact lenses colonized by either S. aureus 8325-4 or the mutant strain. The clinical presentation, bacterial cultures, and histology of the ulceration were examined. RESULTS: Both strains of S. aureus produced similar levels of caseinase, gelatinase, elastase, hyaluronidase, and beta-toxin. S. aureus DU1090 induced weaker haemolysis of rabbit blood cells than S. aureus 8325-4. Ulceration in the S. aureus DU1090 eye was less frequent and less severe than that caused by S. aureus 8325-4. CONCLUSIONS: The enzyme production profile of S. aureus DU1090 was similar to the parent strain. S. aureus strains may produce CLPU-like lesions irrespective of alpha-toxin production, but severe infectious lesions are produced only in the presence of alpha-toxin.

Animals↗
PubMed2005

Interleukin-4 is not critical to pathogenesis in a mouse model of Pseudomonas aeruginosa corneal infection.

PURPOSE: To determine the contribution of interleukin-4 (IL-4) to the initial host response during corneal infection with Pseudomonas aeruginosa in a mouse model. METHODS: Corneas of 6- to 8-week-old IL-4(-/-) and wild-type mice were topically challenged with P. aeruginosa. Ocular tissue was collected 24 hr and 7 days postchallenge. Viable bacterial counts, myeloperoxidase assays, cytokine levels, and clinical and histological examinations were performed. RESULTS: During challenge with P. aeruginosa, no differences were observed clinically, histologically, or in bacterial load between IL-4(-/-) and wild-type mice at either time point. However, differences in cytokine levels of IL-6, KC, and IL-10 were observed. CONCLUSIONS: The data presented indicate that IL-4, a central Th2 cytokine, may not be critical to the pathogenesis or bacterial clearance in this model of P. aeruginosa bacterial keratitis during the early stages of the infectious process.

Animals↗
PubMed2005

A Staphylococcus aureus mouse keratitis topical infection model: cytokine balance in different strains of mice.

Staphylococcus is a leading cause of the potentially blinding disease microbial keratitis. Even with the use of antibiotic therapy, the host inflammatory response continues to damage the cornea, which may lead to blindness. Manipulation of the host response may help improve patient outcome from this devastating disease. We aim to understand the contribution of the host response to Staphylococcus aureus infection. A S. aureus keratitis mouse model was developed in both C57BL/6 and BALB/c mice using two different strains of S. aureus (8325-4 and Staph 38). Twenty-four hours postinfection, mice were killed and eyes were harvested for enumeration of bacteria, polymorphonuclear leucocytes, chemokines and cytokines. The laboratory strain 8325-4 was not as virulent as the clinical isolate Staph 38. In vitro data showed a 250-fold increase in invasion of human corneal epithelial cells by Staph 38 compared to 8325-4. BALB/c mice were susceptible to S. aureus infection whereas C57BL/6 mice were resistant. The resistant C57BL/6 mice were polarized towards a Th2 response, which may be protective for these mice. IL-4, IL-10 and IL-6 were elevated significantly in C57BL/6 mice infected with Staph 38 (P < 0.05). Macrophage inflammatory peptide (MIP)-2 was also significantly elevated in C57BL/6 mice (P < 0.001). The susceptible BALB/c mice had a muted cytokine response, which suggests that S. aureus might be 'walled off' during infection and might avoid host defences. IL-4, IL-10 and IL-6 cytokines may be protective during Gram-positive corneal infection and therefore may be useful for adjunct therapies in the treatment of this disease.

Animals↗
PubMed2004

Pseudomonas aeruginosa with lasI quorum-sensing deficiency during corneal infection.

PURPOSE: To understand the importance of Pseudomonas aeruginosa quorum-sensing systems in the development of corneal infection, the genotypic characteristics and pathogenesis of seven ocular isolates with low-protease and acyl homoserine lactone (AHL) activity and quorum-sensing mutants of PAO1 deficient in lasI, lasR, or rhlR were investigated in the study. METHODS: The possession of the quorum-sensing genes lasI, lasR, rhlI, rhlR, and the quorum-sensing controlled genes lasB, aprA, and rhlAB in the clinical isolates were determined by polymerase chain reaction and Southern blot hybridization. Elastinolytic activity, controlled by the las system, was assayed using elastin Congo red and rhamnolipid production controlled by the rhl system was assessed using agar plates containing methylene blue/cetyltrimethyl ammonium bromide. Induction of keratitis was examined in a scarified inbred BALB/c mouse model. RESULTS: The clinical isolates Paer1 and -3 were lasI and lasR negative, and the isolates Paer2 and -4 were rhlR and rhlAB negative. The isolates Paer17, Paer26, 6294 and 6206 possessed all the genes examined. There was no rhamnolipid production in clinical isolates Paer2 and -4. The isolates Paer1 and -3 were virtually avirulent in the scarified mouse corneas. Using isogenic PAO1 mutants, strain lasI showed a markedly reduced virulence in the corneal infection model. The remainder of the clinical isolates and the lasR or rhlR mutant strains caused severe keratitis. CONCLUSIONS: These results indicate that quorum-sensing deficiency may occur naturally in clinical isolates, and the possession of lasI and hence a functional Las quorum-sensing system may be important in development of corneal infection.

Animals↗
PubMed2004

Secretory phospholipase A2 deposition on contact lenses and its effect on bacterial adhesion.

PURPOSE: Secretory phospholipase A2 (sPLA2) is a potent antibacterial enzyme in tears and has been found to kill Staphylococcus aureus rapidly in vitro. The purpose was to determine whether sPLA2 deposition is associated with contact lens (CL) type, if sPLA2 remains active on CLs, and if this has an effect on bacterial adhesion. METHODS: Ionic (etafilcon A) and nonionic (Polymacon) high-water, soft CLs were used. CLs were worn for 6 hours (daily wear, n = 39) or 6 nights on an extended-wear schedule (n = 25). Tears were collected from patients and worn contact lenses were removed and protein and active enzymes extracted for estimation of their levels. The number of S. aureus adhering to sPLA2-soaked CLs in vitro was also quantified. RESULTS: There was no significant difference in the concentration of sPLA2 in tears between groups of daily CL wearers. Significantly less sPLA2 was recovered from Polymacon CLs for both daily and extended wear compared with etafilcon A CLs (daily wear: 3 vs. 5 ng/lens; extended wear: 3 vs. 6 ng/lens; P < 0.05). sPLA2 activity correlated with protein amounts from lenses. Relatively less active sPLA2 was recovered from Polymacon contact lenses. sPLA2 reduced adhesion of Staphylococcus to contact lenses in vitro. CONCLUSIONS: Etafilcon A CLs absorb more active sPLA2 than Polymacon CLs, which increases with length of CL wear. The sequestering of sPLA2 onto CLs did not affect amounts of the enzyme in tears. sPLA2 adsorbed to a CL can reduce the viable Staphylococcus adhering to the CL, which may protect the eye from colonization by this pathogen.

Absorption↗
PubMed2003

Isolation of conjunctival mucin and differential interaction with Pseudomonas aeruginosa strains of varied pathogenic potential.

The purpose of the study was to investigate the adhesion of Pseudomonas aeruginosa strains with varying pathogenic potential to purified ocular mucin. Bovine conjunctival mucin was purified by three sequential density gradient centrifugation steps. Immobilised mucin was probed with biotin-labelled bacteria isolated from different contact lens events and quantified by densitometry. Bacterial pili were identified by electron microscopy. The results indicate that purified ocular mucin consisted of a polydisperse high molecular weight population containing at least one species of goblet cell origin and was associated with a 97 kDa mucin-associated protein. Three pathogenic P. aeruginosa strains, Paer1 (57.5 +/- 10.8x10(6) CFU ml(-1); contact lens induced acute red eye (CLARE)), 6294 (127.0 +/- 4.7x10(6) CFU ml(-1); microbial keratitis) and Paer25 (60.5 +/- 11.3x10(6) CFU ml(-1); CLARE) exhibited a significantly higher level of adhesion to mucin than the negative control, E. coli (14.3 +/- 9.6x10(6) CFU ml(-1)) (p<0.005). The remaining P. aeruginosa isolates, Paer3 (asymptomatic patient), Paer12 (microbial keratitis) and ATCC 15442 (standard environmental strain) did not significantly differ in their mucin adhesion from the negative control. The majority of bacterial strains tested contained pili; thus differences in mucin adhesion observed could not be solely explained by pili status. In conclusion, P. aeruginosa isolates exhibit differential adhesion patterns to purified ocular mucin. It is proposed that more avid mucin-adhering strains are given the opportunity to adhere and subsequently penetrate the mucous layer of the tear film to initiate pathogenesis.

Animals↗
PubMed2003

The effect of protein-coated contact lenses on the adhesion and viability of gram negative bacteria.

PURPOSE: Gram negative bacterial adhesion to contact lenses can cause adverse responses. During contact lens wear, components of the tear film adsorb to the contact lens. This study aimed to investigate the effect of this conditioning film on the viability of bacteria. METHODS: Bacteria adhered to contact lenses which were either unworn, worn for daily-, extended- or overnight-wear or coated with lactoferrin or lysozyme. Numbers of viable and total cells were estimated. RESULTS: The number of viable attached cells was found to be significantly lower than the total number of cells on worn (50% for strain Paer1 on daily-wear lenses) or lactoferrin-coated lenses (56% for strain Paer1). Lysozyme-coated lenses no statistically significant effect on adhesion. DISCUSSION: The conditioning film gained through wear may not inhibit bacterial adhesion, but may act adversely upon those bacteria that succeed in attaching.

Anti-Infective Agents↗
PubMed2003

Clinical characterization of corneal infiltrative events observed with soft contact lens wear.

PURPOSE: Corneal infiltrates are commonly observed during adverse reactions associated with contact lens wear. Broad ranges of presentations are encountered, and there is no well-established classification system. The aim of this paper is to categorize corneal infiltrates associated with soft lens wear and present the typical clinical characteristics associated with each type of event. METHODS: All events of corneal infiltrates occurring in soft contact lens clinical trials over 10 years (9 years retrospective and 1 year prospective) and conducted at two contact lens research centers were reviewed by a panel of experts comprising ophthalmologists, optometrists, and other biologic scientists. Classification of each event was based on assessment of a range of signs and symptoms by the review panel. RESULTS: A classification scheme was devised to distinguish infiltrative events that were serious and symptomatic (microbial keratitis), clinically significant and symptomatic (contact lens-induced peripheral ulcer, contact lens-induced acute red eye, infiltrative keratitis), and clinically nonsignificant and asymptomatic (asymptomatic infiltrative keratitis and asymptomatic infiltrates). CONCLUSION: Corneal infiltrates can be classified into six distinct categories. This classification scheme, based on clinical characteristics, should aid diagnosis, management, and treatment of corneal infiltrates as well as assisting investigations into the etiology of each of these conditions.

Contact Lenses, Hydrophilic↗
PubMed2003

Evasion of cellular ocular defenses by contact lens isolates of Serratia marcescens.

PURPOSE: Contact lens contamination by Serratia marcescens can lead to the development of microbial keratitis and contact lens-induced acute red eye, particularly during overnight contact lens wear. The aim of this study was to investigate the interaction of S. marcescens with polymorphonuclear leukocytes (PMNs), which occur in closed-eye tears. METHODS: The respiratory burst of PMNs, when incubated with S. marcescens, was measured using chemiluminescence. Phagocytosis of planktonic and biofilm-grown bacteria by PMNs was also determined. RESULTS: Some strains of S. marcescens did not induce a respiratory burst from PMNs and resisted phagocytosis regardless of opsonization. Phagocytic resistance and growth in the presence of PMNs was markedly increased when bacteria were grown as a biofilm on a contact lens. CONCLUSIONS: The ability of S. marcescens to resist phagocytosis and grow to high levels in the presence of PMNs, particularly when grown as a biofilm on contact lenses, may be a mechanism by which this bacterium can survive the ocular host defense system, grow, and cause keratitis or other adverse responses.

Bacteriological Techniques↗
PubMed2003

Mucin degradation mechanisms by distinct Pseudomonas aeruginosa isolates in vitro.

Pseudomonas aeruginosa has emerged as an important causative agent of bacterial keratitis, a rapidly progressive ocular condition that may result in blindness. Secretory mucin forms the main constituent of the precorneal tear film, a three-layer film on the ocular surface protecting the underlying corneal epithelium from potential pathogens. The purpose of the present study was to compare mucin degradation mechanisms between ocular P. aeruginosa strains. Mucin degradation was assessed by agarose electrophoresis, lectin blotting, and size exclusion chromatography. The results indicate that certain P. aeruginosa strains (Paer12, ATCC 15442, 6294, and Paer25) had depleted mucin from the culture supernatant and that this was contingent on the inherent ability of these isolates to produce proteases. Non-protease-producing strains (Paer1 and Paer3) did not appreciably degrade mucin. Further, galactosidase, N-acetylglucosaminidase, and N-acetylgalactosaminidase activities were detected in some strains, suggesting the operation of further mechanisms of mucin degradation by P. aeruginosa. Mucin degradation by P. aeruginosa also seemed to be for the acquisition of nutrients, as a growth advantage was observed in mucin-depleting strains over nondepleting strains in the long term. It is postulated that the degradation of mucin serves to collapse the mucin barrier and its associated network containing antibacterial tear components and to provide energy for sustained bacterial growth.

Chromatography, Gel↗
PubMed2003

Pseudomonas aeruginosa keratitis in IL-6-deficient mice.

BACKGROUND: Pseudomonas aeruginosa infection is one of the most destructive diseases of the eye. The host response to this infection is critical to the outcome. Interleukin-6 (IL-6) is implicated in this response; however, the mechanisms by which IL-6 contributes to the host defences in corneal infection remain unclear. Using IL-6-/- mice, we have explored the role of IL-6 in P. aeruginosa keratitis. METHODS: The eyes of IL-6 gene knockout and wild-type mice were challenged topically with P. aeruginosa and examined on days 1-7. Keratitis was examined clinically and histologically. Cytokine, chemokine and complement 3 levels were determined by ELISA and ICAM-1 by immunohistochemistry. RESULTS: Clinically, the IL-6-/- mice showed more severe disease than wild-type mice and this was supported by the histological findings. More than 2-fold higher bacterial load was detected in the eyes of the IL-6-/- mice than in those of the wild-type mice. Neutrophil infiltration to the central cornea of the IL-6-/- mice failed to occur in response to infection, although a greater number of neutrophils were present in the whole eye. This may in part be due to the reduced expression of the adhesion molecule ICAM-1 in the cornea, but does not appear to stem from insufficient production of chemokines or complement 3. CONCLUSIONS: Our findings indicate that IL-6 is critical to the host defence of the cornea during P. aeruginosa infection. Pharmacological manipulation of the IL-6 response may represent a rational strategy for new interventions.

Animals↗
PubMed2003

Regulation of MMPs and TIMPs by IL-1beta during corneal ulceration and infection.

PURPOSE: This study was conducted to investigate the role of IL-1beta in the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in a mouse model of experimental keratitis and corneal injury. METHODS: Mice were injected subconjunctivally with 10 micro g of anti-mouse IL-1beta antibody 2 hours before challenge with Pseudomonas aeruginosa (strain 6294). Control animals received an equal volume and concentration of isotype control antibody at the same time. Eyes were enucleated at 0, 8, 24, and 72 hours, after bacterial challenge and processed for histologic examination. Some eyes were homogenized and used to evaluate production of MMP-2, MMP-9, TIMP-1, and TIMP-2 protein, by zymography and reverse zymography. RESULTS: Injury without bacterial infection resulted in increases in both MMP-2 and -9 and a slight but significant downregulation of TIMP-1. Administration of anti-IL-1beta just before injury and without bacterial infection resulted in a significant reduction in expression of MMP-2 (at 8 hours), MMP-9 (at 8 hours), TIMP-1 (at 8 and 72 hours), and TIMP-2 (at 8 hours). Mice treated with anti-IL-1beta antibody, before bacterial challenge, demonstrated markedly reduced corneal damage compared with the severe corneal injury and massive neutrophil infiltration observed in infected mice treated with control antibody. Administration of the neutralizing anti-IL-1beta antibody resulted in a significant reduction of MMP-9 and a change in the time course of TIMP-1 and -2 expression. The reduction in MMP-9 by anti-IL-1beta during infection was much greater than the reduction without infection. CONCLUSIONS: The results imply that IL-1beta has a central role in corneal destruction during bacterial keratitis and suggests that targeting IL-1beta may be a novel therapeutic strategy for microbial keratitis.

Animals↗