Biomedical subjects

M D Willcox

Publications and source records attributed to M D Willcox.

At least 19 recordsLinked to original sources

PubMed2001

Bacterial interactions with contact lenses; effects of lens material, lens wear and microbial physiology.

Contact lens wear is a successful form of vision correction. However, adverse responses can occur during wear. Many of these adverse responses are produced as a consequence of bacterial colonization of the lens. The present study demonstrated that during asymptomatic contact lens wear lenses are colonized by low levels of bacteria with gram-positive bacteria, such as coagulase negative staphylococci, predominating. Gram-negative bacteria are frequently the causative agents of adverse responses during contact lens wear. Measuring the adhesion of different strains and/or species of bacteria to different contact lens materials demonstrated considerable differences. In particular. Pseudormonas aeruginosa strains Paerl and 6294 and Aeromonas hydrophilia strain Ahyd003 adhered in larger numbers to the highly oxygen permeable contact lenses Balafilcon A compared to hydrogel lenses manufactured from either Etafilcon A or HEMA. Furthermore, after Balafilcon A lenses had been worn for 6 h during the day bacteria were able to adhere in greater numbers to the worn lenses compared to the unworn lenses with increases in adhesion ranging from 243% to 1393%. However, wearing Etafilcon A lenses usually resulted in a decrease in adhesion (22-48%). Bacteria were able to grow after adhesion to lenses soaked in artificial tear fluid and formed biofilms, visualized by scanning confocal microscopy. Chemostat grown bacterial cultures were utilized to enable control of bacterial growth conditions and bacteria were shown to adhere in the greatest numbers if grown under low temperature (25 degrees C compared to 37 degrees C). The changes in growth temperature was shown. using 2D gel electrophoresis, to change the experssion of cell-surface proteins and, using ID gel electrophoresis, to change the expression of surface lipopolysaccharide of P. aeruginosa Paerl. Thus, these surface changes would have been likely to have mediated the increased adhesion to Etafilcon A contact lenses.

Aeromonas hydrophila↗

PubMed2001

Lactoferrin-induced reduction of vanB vancomycin resistance in enterococci.

The mucosal protein lactoferrin (LF) reduces the MIC of vancomycin for staphylococcal isolates sensitive to this glycopeptide. The purpose of this research was to investigate the effect of LF on the MIC of vancomycin for vanB resistant isolates of Enterococcus faecalis (Efs1) and E. faecium (Efm1). At a concentration of 2048 microg/ml, LF reduced the MIC of vancomycin 16-fold for Efs1 and eight-fold for Efm1. The cell wall precursors of Efs1 were examined following growth in media supplemented with LF, D-alanine or D-lactate. The precursors were extracted from harvested cells by ether and ion exchange chromatography and the amino acid and lactate composition was determined. Compared with that of unsupplemented media, D-alanine or LF supplementation caused an increase in the D-alanine content of the precursors. Concomitantly, the D-lactate content was reduced. Exogenous D-lactate did not affect the composition of the precursors. This suggests that LF caused an increase in the pool of pentapeptide cell wall precursors. The LF-induced reduction in the vancomycin resistance of enterococci at in vivo concentrations suggests a potential use for this protein as an adjunctive agent to vancomycin.

Cell Wall↗

PubMed2001

Contact lens related corneal infections.

This article describes microbial keratitis, infection of the cornea by micro-organisms. Contact lens wear is a predisposing factor for the development of microbial keratitis. Micro-organisms probably adhere to the contact lens, transfer from the contact lens to a damaged or compromised corneal epithelial surface, penetrate into the deeper layers of the cornea and produce corneal damage. Host responses to the invading micro-organisms, while designed to protect the eye, can often exacerbate the situation and the resulting microbial keratitis can lead to permanent blindness. The microbial, biochemical and immunological aspects of MK will be described in detail.

Anti-Bacterial Agents↗

PubMed2001

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 signifcantly 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.

Bacterial Adhesion↗

PubMed2001

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.

Bacterial Adhesion↗

PubMed2001

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.

4-Butyrolactone↗

PubMed2001

Random amplified polymorphic DNA analysis of Acinetobacter species isolated from worn contact lenses.

The purpose of this study was to explore the genotypes of Acinetobacter species and to compare the genotypes and phenotypes of the Acinetobacter isolated from contact lens wearers. Nineteen strains were used in the study, 13 were isolated from patients experiencing an adverse response event, and six strains were isolated from asymptomatic contact lens wearers. Random amplified polymorphic DNA and phenotypic analyses were carried out using commercially available kits. Random amplified polymorphic DNA analysis showed a higher discrimination power compared with the phenotypic analysis. The test strains were loosely clustered into six genotypic groups with no definite relation to any clinical events. These results indicate that many genotypes of Acinetobacter can cause adverse responses, and the initial source of the microorganisms rather than their clinical sequelae may determine classification.

Acinetobacter↗

PubMed2001

Expression of macrophage migration inhibitory factor during Pseudomonas keratitis.

Macrophage migration Inhibitory factor (MIF) is a recently rediscovered pro-inflammatory cytokine, and has been shown to play a role in the regulation of neutrophil chemokines and angiogenesis. Corneal epithelial and endothelial cells have been shown to express MIF. This study evaluated the expression of MIF during Pseudomonas keratitis in mice and in vitro using a corneal epithelial cell line. Three strains of P. aeruginosa, 6294 (invasive strain), 6206 (cytotoxic strain) and Paer1 (non-infectious strain) were used. Both cytotoxic and invasive strains were isolated from human corneal ulcers and the Paer1 strain was isolated from a non-infectious condition. Following challenge in mouse corneas or a corneal epithelial cell line, corneal homogenates or lysed corneal epithelial cells were used to isolate RNA. Migration inhibitory factor mRNA expression in the mouse cornea or human corneal epithelial cells was examined by reverse transcription-polymerase chain reaction analysis, and was found to be expressed as early as 4 h after the injury (scratch controls) or infection in the mouse corneas. Migration inhibitory factor mRNA in scratch controls and Paer1-inoculated corneas showed peak levels at 4 h post-challenge and this dropped by 24 h post-challenge. Corneas challenged with invasive and cytotoxic strains showed peak expression 24 h post-challenge. Migration inhibitory factor mRNA levels were significantly higher in invasive and cytotoxic strain inoculated corneas compared to Paer1 inoculated corneas. Challenging the corneal epithelial cell line with Pseudomonas 6294 and 6206 strains induced peak expression at 8 h and levels were decreased by 12 h. Epithelial cells inoculated with recombinant human interleukin-1beta protein induced very high levels of MIF mRNA at all time points compared to infected and control corneal epithelial cells. High expression of MIF in the infected corneas suggests that it may have a role in the pathogenesis of corneal disease induced by invasive and cytotoxic strains of P. aeruginosa.

Animals↗

PubMed2001

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 effectve 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.

Anti-Bacterial Agents↗

PubMed2001

Lacryglobin in human tears, a potential marker for cancer.

Lacrygobin has been identified in human tears. This protein has high sequence homology to the mammaglobins, proteins upregulated in breast cancer and in breast cancer metastasis. In order to investigate the utility of tear screening for cancer tear samples were collected from patients with different types of cancer and compared to controls. Tear samples were taken from five controls and eight breast, six lung, five colon, one prostate and three ovary cancer patients. Tears were analysed using 2-D gel electrophoresis (n = 25) and 1-D electrophoresis (n = 3). Lacryglobin was present in the following percentage of patients: breast cancer (88%), lung (83%), colon (100%), ovary (33%), prostate (100%) and controls (60%). Two control patients with lacryglobin had a family history of breast and prostate cancer. Lacryglobin was detected in some but not all tear samples and further studies are warranted to investigate its potential as a marker for cancer.

Adult↗

PubMed2001

Regulatory role of IL-1beta 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)-1beta and macrophage inflammatory protein-2 are associated with the severity of corneal infection. Interleukin-1beta is a principal inflammatory mediator. Understanding the regulatory role of IL-1beta 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-1beta antibody (IL-1beta mAb). The culture supernatants were collected for measuring cytotoxicity and protein levels of IL-1beta, IL-8 and IL-6 by enzyme-linked immunosorbent assay. Results showed that HCE cells expressed low levels of IL-1beta and high levels of IL-6 and IL-8 during P. aeruginosa colonization. Paer1-colonized HCE cells produced higher levels of IL-1beta, IL-6 and IL-8 protein compared to those produced by 6206- and 6294-colonized HCE cells. Administration of IL-1beta mAb decreased the production of IL-8 and IL-6. In conclusion, P. aeruginosa-colonized HCE cells produced low levels of IL-1beta and high levels of IL-6 and IL-8. Neutralizing IL-1beta protein significantly downregulated the production of IL-8 and IL-6.

Cell Line↗

PubMed2001

Association of acinetobacter species with contact lens-induced adverse responses.

PURPOSE: To determine the levels of Acinetobacter species associated with normal soft contact lens wear and to determine whether Acinetobacter species are involved in adverse reactions that occur during contact lens wear. METHODS: Patients wore soft contact lenses on an extended-wear basis. The bacteria on lenses and ocular swabs during asymptomatic and symptomatic lens wear were identified using standard microbiologic methods. RESULTS: Acinetobacter species were isolated and identified from 16 (13%) of 126 patient samples. Greater numbers of Acinetobacter species were isolated from lenses of patients experiencing adverse responses than from asymptomatic patients. Acinetobacter species were isolated from patients experiencing symptomatic adverse responses in 4 (13%) of 32 cases. CONCLUSION: It appeared that Acinetobacter species colonized the eye of extended contact lens wearers at a time when the normal functioning of the eye was compromised by contact lens wear. When Acinetobacter species were in high numbers on a contact lens, an adverse response occurred. This may implicate Acinetobacter species as a contributing factor to adverse responses associated with contact lens wear.

Acinetobacter↗

PubMed2001

Microbial colonization of soft contact lenses over time.

PURPOSE: To compare the bacterial colonization of soft contact lenses in subjects for successively increasing periods, up to 13 nights of wear. The aim of this study was to determine whether increasing the length of lens wear predisposed subjects to high levels of microbial colonization of lenses. METHODS: Subjects (N = 20) were divided into those with a prior history of adverse events (N = 6), gram-negative bacterial carriers (N = 6), and those with no previous history (N = 8). RESULTS: There were no temporal changes in microbial colonization of lenses. Lenses from all wearers were colonized at least once during the study by gram-positive bacteria at low numbers (<10 cfu/ml). Gram-negative bacteria colonized lenses at least once in 80% of all wearers. Lenses from gram-negative bacterial carriers were more frequently colonized by Staphylococcus aureus and Pseudomonas sp. compared with subjects with no previous history and subjects with a prior history of adverse events, respectively. Lenses from gram-negative bacterial carriers were less frequently colonized by a range of gram-positive bacteria compared with subjects with a prior history of adverse events. CONCLUSIONS: Increasing the length of lens wear up to 13 nights did not result in a predictable increase in bacterial colonization of contact lenses. Gram-positive bacteria were isolated frequently but in low numbers, whereas gram-negative bacteria were present sporadically.

Adult↗

PubMed2001

Streptococcus australis sp. nov., a novel oral streptococcus.

Strains of streptococci were isolated from the mouths of children attending the United Dental Hospital, Sydney, Australia. These strains were analysed biochemically using the Rapid ID32 Strep microsystem, were subjected to DNA-DNA hybridization with other members of the oral streptococci and had their 165 rRNA analysed. On the basis of DNA-DNA hybridization, their nearest relative was Streptococcus parasanguinis, whereas, on the basis of 16S rRNA analysis, it was Streptococcus infantis. The name Streptococcus australis sp. nov. is proposed for the new species. The type strain is AI-1T (= ATCC 700641T = NCTC 13166T).

Base Composition↗

PubMed2001

Effector mechanisms of protection against Pseudomonas aeruginosa keratitis in immunized rats.

Pseudomonas aeruginosa is an opportunistic pathogen which causes sight-threatening corneal infections in humans. The purpose of this study was to evaluate various immunization routes that may provide protection against Pseudomonas keratitis and to define the molecular mechanisms involved in the protection. Sprague-Dawley rats (10 to 12 weeks old) were immunized using paraformaldehyde-killed P. aeruginosa (strain 6206) via oral, nasal, and intra-Peyer's patch (IPP) routes followed by an ocular topical booster dose. Scratched corneas were challenged with an infective dose of P. aeruginosa. Following clinical examination, eyes were enucleated for histology, polymorphonuclear leukocyte (PMN) quantitation, bacterial count, enzyme-linked immunosorbent assay, and RNase protection assay. PMN infiltration was higher early (4 h) during the infection in immunized rats than in nonimmunized rats. Later during the infection, the number of PMNs diminished in immunized rats while in nonimmunized animals the number of PMNs continued to increase. Bacteria were cleared much faster from immunized groups than from the nonimmunized group, and the nasally immunized group had the most efficacious response among the immunized groups. Nasal and IPP immunization groups had increased cytokine expression of interleukin-2 (IL-2) and IL-5 and differed from each other for IL-6. All three immunized groups had significantly reduced IL-1 beta levels when compared with the nonimmunized rats and a significantly altered profile for CINC-1 expression. This study has shown that the route of immunization modulates the inflammatory response to ocular P. aeruginosa infection, thus affecting the severity of keratitis and adverse pathology, with nasal immunization being the most effective.

Administration, Intranasal↗

PubMed2001

Bacterial populations on 30-night extended wear silicone hydrogel lenses.

PURPOSE: Ocular infection and inflammation during hydrogel lens extended wear is often associated with colonization of the lenses with bacteria. This study compares colonization of a high Dk silicone hydrogel contact lens (lotrafilcon A) worn on a 30-night extended wear basis to a low Dk HEMA-based lens (etafilcon A) worn on a 6-night extended wear schedule. METHODS: The group wearing the low Dk/t soft contact lens (n = 63) replaced their lenses weekly and the group wearing high Dk/t soft contact lenses replaced their lenses monthly (n = 64). Lens allocation was assigned randomly at enrollment. Worn lenses, from one eye only, were collected aseptically and placed in sterile vials. Microbial growth on various media was enumerated and the number of colony forming units (cfu) per lens was calculated in categories of normal ocular microbiota (such as coagulase-negative staphylococci and Propionibacterium spp.) and known ocular pathogens (such as Staphylococcus aureus and gram-negative bacteria). The proportion of samples colonized with these bacteria and the extent of colonization were compared between the two groups. The proportion of sterile lenses was calculated, and the types of bacteria on each lens group were compared. RESULTS: No differences between the low and high Dk/t Soft contact lens groups were observed in the proportion of lenses colonized by Propionibacterium spp. (48% vs 43%, P = 0.4) or coagulase-negative staphylococci (47% vs 54%, P = 0.2). Similarly, no differences were found for lenses colonized by S. aureus (0% vs 2%, P = 0.1) or gram-negative bacteria (3% vs 2%, P = 0.8). The types of bacteria isolated from the high and low Dk/t lenses were similar. There were no differences in the number of sterile samples (28% vs 27%, P = 0.8) from each group. CONCLUSIONS: These findings suggest that high Dk/t silicone hydrogel materials are colonized by similar numbers and types of microorganisms during extended wear compared to HEMA-based material. Most lenses were colonized by commensal bacteria during 30-night extended wear with high Dk/t lenses and during 6-night extended wear with low Dk/t lenses. The incidence of sterile samples was the same between the high and low Dk/t soft contact lens groups.

Adult↗

PubMed2001

Staphylococcus corneal virulence in a new topical model of infection.

PURPOSE: To develop a topical inoculation model of Staphylococcus aureus keratitis in which scarification, contact lenses, and spermidine are used to inhibit the host defenses and to investigate the role of alpha-toxin in this infection. METHODS: An alpha-toxin-positive parent strain (8325-4), its isogenic alpha-toxin-negative mutant (DU1090), and a genetically rescued form of the mutant (DU1090/pDU1212) were bound to rabbit-specific contact lenses, treated with spermidine (50 mM), and applied to scarified rabbit corneas. Eyes were treated topically with spermidine before and after lens application. Eyes were graded for disease by slit lamp examination (SLE) every 6 hours until 24 hours PI (PI), and erosion diameters were measured. Histopathologic changes and colony forming units (CFUs) of bacteria were determined. RESULTS: Spermidine treatment and inoculation of eyes with Staphylococcus on contact lenses resulted in significant increases in both CFUs per cornea (P = 0.0041) and SLE score (P or= 0.1959) multilog increase in CFUs over the inoculum at 24 hours PI. The alpha-toxin-producing strains, 8325-4 and DU1090/pDU1212, caused significantly more disease than the alpha-toxin-deficient mutant DU1090 at 24 hours PI (P <or= 0.0001). Histopathology revealed bacteria in scarified regions of the corneas and, for 8325-4 and DU1090/pDU1212, extensive epithelial sloughing and severe inflammation. CONCLUSIONS: A new topical model of infection has been developed, and alpha-toxin is an important virulence factor in this model.

Animals↗

PubMed2000

Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1.

With the completion of many genome projects, a shift is now occurring from the acquisition of gene sequence to understanding the role and context of gene products within the genome. The opportunistic pathogen Pseudomonas aeruginosa is one organism for which a genome sequence is now available, including the annotation of open reading frames (ORFs). However, approximately one third of the ORFs are as yet undefined in function. Proteomics can complement genomics, by characterising gene products and their response to a variety of biological and environmental influences. In this study we have established the first two-dimensional gel electrophoresis reference map of proteins from the membrane fraction of P. aeruginosa strain PA01. A total of 189 proteins have been identified and correlated with 104 genes from the P. aeruginosa genome. Annotated membrane proteins could be grouped into three distinct categories: (i) those with functions previously characterised in P. aeruginosa (38%); (ii) those with significant sequence similarity to proteins with assigned function or hypothetical proteins in other organisms (46%); and (iii) those with unknown function (16%). Transmembrane prediction algorithms showed that each identified protein sequence contained at least one membrane-spanning region. Furthermore, the current methodology used to isolate the membrane fraction was shown to be highly specific since no contaminating cytosolic proteins were characterised. Preliminary analysis showed that at least 15 gel spots may be glycosylated in vivo, including three proteins that have not previously been functionally characterised. The reference map of membrane proteins from this organism is now the basis for determining surface molecules associated with antibiotic resistance and efflux, cell-cell signalling and pathogen-host interactions in a variety of P. aeruginosa strains.

Bacterial Proteins↗