The structure and function of modular Escherichia coli O157:H7 bacteriophage FTBEc1 endolysin, LysT84: defining a new endolysin catalytic subfamily.

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level - key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the ß(1-4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.

Differences in sphere-forming cells from keratoconic and normal corneal tissue: Implications for keratoconus pathogenesis.

Keratoconus is primarily an anterior corneal disorder of unclear aetiology. Stem cells may play a role in the perpetuation of keratoconus, although this has yet to be definitively established. Sphere-forming cells from normal human donor corneas have previously been shown to be a heterogenous mix of epithelial, stromal, stem and progenitor cell components which have potential for treatment of corneal dystrophies. Our work set out to isolate and characterise sphere-forming cells from human keratoconic tissue. Keratoconic donor corneas were successfully used to culture sphere-forming cells in vitro. Time lapse imaging of these spheres on a collagen surface over 8 days revealed keratoconic spheres lack the ability to maintain a central core and have diminished ability to repopulate the surface. Immunocytochemistry showed positive labelling for the stem cell marker 'Adenosine triphosphate-binding cassette sub-family B member 5 (ABCB5)' indicating stem cell retention and the myofibroblast marker alpha smooth muscle actin indicating wound repair while droplet digital Polymerase Chain Reaction confirmed an increase in expression of stem and stromal cell markers in keratoconic spheres compared to spheres cultured from normal donors at day 7 post-placement. Keratoconic sphere-forming cells showed a diminished repopulation ability, a faster wound healing response and lack of central core retention. These results suggest stem cells in keratoconus may be in an elevated state of wound repair and unable to respond appropriately to further injury in corneal maintenance. Sphere forming cell populations in keratoconus appear to be different to those isolated from normal corneas and this may be an important consideration in unearthing keratoconus aetiology.

Sphere-forming cells from peripheral cornea demonstrate a wound-healing response to injury.

The cornea is the initial refractive interface of the eye. Its transparency is critical for clear vision and is maintained by stem cells which also act to repair injury inflicted by external insults, such as chemical and thermal burns. Damage to the epithelium compromises its clarity and can reduce or eliminate the stem cell population, diminishing the ability for self-repair. This condition has been termed "limbal stem cell deficiency"; severe cases can lead to corneal blindness. Sphere-forming cells isolated from peripheral cornea are a potential source of stem and progenitor cells for corneal repair. When provided with appropriate substrate, these spheres have the ability to adhere and for cells to migrate outwards akin to that of their natural environment. Direct compression injury and remote scratch injury experiments were conducted on the sphere cells to gauge their wound healing capacity. Measures of proliferation, differentiation, and migration were assessed by immunohistochemical detection of EdU incorporation, α-smooth muscle actin expression and confocal image analysis, respectively. Both modes of injury were observed to draw responses from the spheres indicating wound healing processes. Direct wounding induced a rapid, but transient increase in expression of α-SMA, a marker of corneal myofibroblasts, followed by a proliferative and increasing migratory response. The spheres were observed to respond to remote injury as entire units, with no directional response seen for targeted repair over the scratch injury area. These results give strength to the future use of these peripheral corneal spheres as transplantable units for the regeneration of corneal tissue.

Spectrum of bacterial colonization associated with urothelial cells from patients with chronic lower urinary tract symptoms.

Chronic lower urinary tract symptoms (LUTS), such as urgency and incontinence, are common, especially among the elderly, but their etiology is often obscure. Recent studies of acute urinary tract infections implicated invasion by Escherichia coli into the cytoplasm of urothelial cells, with persistence of long-term bacterial reservoirs, but the role of infection in chronic LUTS is unknown. We conducted a large prospective study with eligible patients with LUTS and controls over a 3-year period, comparing routine urine cultures of planktonic bacteria with cultures of shed urothelial cells concentrated in centrifuged urinary sediments. This comparison revealed large numbers of bacteria undetected by routine cultures. Next, we typed the bacterial species cultured from patient and control sediments under both aerobic and anaerobic conditions, and we found that the two groups had complex but significantly distinct profiles of bacteria associated with their shed bladder epithelial cells. Strikingly, E. coli, the organism most responsible for acute urinary tract infections, was not the only or even the main offending pathogen in this more-chronic condition. Antibiotic protection assays with shed patient cells and in vitro infection studies using patient-derived strains in cell culture suggested that LUTS-associated bacteria are within or extremely closely associated with shed epithelial cells, which explains how routine cultures might fail to detect them. These data have strong implications for the need to rethink our common diagnoses and treatments of chronic urinary tract symptoms.

Sphere-forming cells from peripheral cornea demonstrate polarity and directed cell migration.

Sphere-forming cells from peripheral cornea represent a potential source of progenitor cells for treatment of corneal degenerative diseases. Control of cellular repopulation on transplantable substrates is important to prevent uncontrolled growth in unfavourable directions. The coordination of cellular outgrowth may be in response to environmental cues and/or cellular signals from other spheres. To investigate this, cell migration patterns were observed following placement of spheres on an adhesive surface. Human peripheral corneal cells were maintained using a sphere-forming assay and their behaviour on collagen substrate recorded by time-lapse imaging. Immunocytochemistry and proliferation assays were used to detect protein expression and cell division. Proliferation assays showed that spheres formed by a combination of cell division and aggregation. Cell division continued within spheres for up to 4 months and was up-regulated when exposed to differentiation medium and collagen substrate. The spheres expressed both epithelial and stromal cell markers. When exposed to collagen; (1) 25% of the spheres showed spontaneous polarised outgrowth. (2) One sphere initially showed polarised outgrowth followed by collective migration with discrete morphological changes to form leading and trailing compartments. (3) A sphere which did not show polarised outgrowth was also capable of collective migration using cell protrusion and retraction. (4) Active recruitment of cells into spheres was observed. (5) Placement of spheres in close proximity led to production of a cell exclusion area adjacent to spheres. Thus peripheral corneal cell spheres are dynamic entities capable of developing polarity and modifying migration in response to their environment.

Lipoinjection for Unilateral Vocal Fold Paralysis Treatment: A Systematic Review and Meta-Analysis.

INTRODUCTION: Lipoinjection is one of the available treatments for unilateral vocal fold paralysis. OBJECTIVE: To evaluate lipoinjection predictability, and analyze the differences in safety and efficacy of the different techniques. STUDY DESIGN: Systematic review and meta-analysis. METHODS AND RESULTS: A systematic review on Medline, Cochrane, and Scopus databases included 49 articles analyzing the data of 1,166 patients, concerning technical details and voice parameters changes. Lipoinjection used a mean volume of 1.3 mL, 95% confidence interval (CI) (0.92, 1.69)-average overcorrection of 30%. Meta-analysis of pre- and postoperative voice parameters' means showed a significant improvement at 6 months of mean phonation time (preoperative: 5.12, 95% CI [4.48, 5.76]-6 months: 10.46, 95% CI [9.18, 11.75]), Jitter (preoperative: 2.71, 95% CI [2.08, 3.33])-6 months: 1.37, 95% CI [1.05, 1.70]), Shimmer (preoperative: 4.55, 95% CI [3.04, 6.07]-6 months: 2.57, 95% CI [1.69, 3.45]), grade (preoperative: 2.15, 95% CI [1.73, 2.57]-6 months: 0.12, 95% CI [0.97, 1.43]), breathiness (preoperative: 2.012, 95% CI [1.48, 2.55]-6 months: 0.99, 95% CI [0.58, 1.40]), and asthenia (preoperative: 1.90, 95% CI [1.33, 2.47]-6 months: 0.75, 95% CI [0.17, 1.33]) of GRBAS (Grade, Roughness, Breathiness, Asthenia and Strain), and Voice Handicap Index-30 (preoperative: 72.06, 95% CI [54.35, 89.76]-6 months: 26.24, 95% CI [19.58, 32.90]). Subgroup analysis by harvesting technique concluded in no statistically significant difference between them. Few complications were reported. Reintervention was only required for 86 patients. CONCLUSION: Lipoinjection seems a safe therapeutic option for unilateral vocal fold paralysis, with available data showing an efficacy lasting 6 months to 1 year. Laryngoscope, 132:1630-1640, 2022.

Randomized controlled trial of toothbrushing to reduce ventilator-associated pneumonia pathogens and dental plaque in a critical care unit.

AIM: To investigate the effect of a powered toothbrush on colonization of dental plaque by ventilator-associated pneumonia (VAP)-associated organisms and dental plaque removal. MATERIALS AND METHODS: Parallel-arm, single-centre, examiner- and analyst-masked randomized controlled trial. Forty-six adults were recruited within 48 h of admission. Test intervention: powered toothbrush, control intervention: sponge toothette, both used four times per day for 2 min. Groups received 20 ml, 0.2% chlorhexidine mouthwash at each time point. RESULTS: The results showed a low prevalence of respiratory pathogens throughout with no statistically significant differences between groups. A highly statistically significantly greater reduction in dental plaque was produced by the powered toothbrush compared with the control treatment; mean plaque index at day 5, powered toothbrush 0.75 [95% confidence interval (CI) 0.53, 1.00], sponge toothette 1.35 (95% CI 0.95, 1.74), p=0.006. Total bacterial viable count was also highly statistically significantly lower in the test group at day 5; Log(10) mean total bacterial counts: powered toothbrush 5.12 (95% CI 4.60, 5.63), sponge toothette 6.61 (95% CI 5.93, 7.28), p=0.002. CONCLUSIONS: Powered toothbrushes are highly effective for plaque removal in intubated patients in a critical unit and should be tested for their potential to reduce VAP incidence and health complications.

Sphere-forming corneal cells repopulate dystrophic keratoconic stroma: Implications for potential therapy.

BACKGROUND: Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss. Cells extracted from peripheral corneas can form stem cell-enriched spheres, which have shown the potential to repopulate the normal peripheral corneal stroma in vitro upon sphere implantation but have not been previously studied in keratoconic tissue. AIM: To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic tissue. METHODS: Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal cells. These spheres were implanted into incisions created in full thickness and onto the surface of 10 µm thin sections of keratoconic and normal stromal tissues in vitro. Tissue sections were used to maximise use of limited keratoconic tissue available for research. Living cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d (D14) from implantation. Sphere cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital PCR to assess the potential implications of these characteristics in the use of spheres in keratoconus treatment. RESULTS: Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10 µm thin en face tissue sections. No observable differences were seen in sphere migration, proliferation or differentiation in keratoconic tissue compared to normal between day 0 and D14. Spheres stained positively with Calcein-AM up to D14. Cell migration increased from day 0 to D14, occurring radially in three dimensions from the sphere and in alignment with tissue edges. Cell proliferation marker, EdU, was detected at day 10. Implanted spheres stained positively for putative stem cell markers ∆Np63α and ABCB5, while ABCG2, ABCB5, ∆Np63 and p63α were detectable by droplet digital PCR up to D14. Double immunolabelling revealed absence of ABCB5 staining in migrated cells but positive staining of alpha smooth muscle actin (myofibroblast marker) in some migrated cells. Droplet digital PCR showed similar expression patterns of differentiation markers but a reduction in stem cell markers between normal and keratoconic tissue with an increase in stromal cell markers and a reduction in epithelial cell markers, indicating an appropriate response to repopulating diseased tissue. CONCLUSION: Cells from implanted stem cell-enriched spheres can repopulate a keratoconic corneal stromal surface in a directed manner and exhibit migratory stromal cell phenotypes.

Molecular evidence for the role of inflammation in dry eye disease.

Ocular surface inflammation is propagated by a complex series of molecular processes and has been implicated in the pathogenesis of dry eye disease (DED), either as a causal or a downstream effect of ocular surface disease. A state of hyperosmolarity elicits an acute immune response in DED, leading to subsequent activation of the adaptive immune response. This cascade incites dysregulation of the immune system, triggering a vicious cycle of events that causes damage to the ocular surface. Symptoms associated with these events include burning, irritation, redness, photophobia and blurred vision. The chronic nature of the disease process can cause permanent alterations to the ocular surface and adnexa. An increasing investment in treatment options, and positive outcomes with novel therapies that have received subsequent regulatory approval, lends further support to the role of inflammation in DED. This review highlights the nature and function of a range of fundamental inflammatory molecules in DED to provide the clinician with an appreciation for the ways in which these factors might be manipulated in DED management.

Use of a Purpose-Built Impression Cytology Device for Gene Expression Quantification at the Ocular Surface Using Quantitative PCR and Droplet Digital PCR.

PURPOSE: To describe an impression cytology (IC) technique using a purpose-built, sterile, EYEPRIM IC device that can be coupled with a TRIzol reagent-based RNA extraction protocol to yield sufficient RNA for gene expression analysis. METHODS: IC samples using the EYEPRIM device were collected from the bulbar conjunctiva, with and without topical anesthesia, and evaluated for RNA yield, the absence of polymerase chain reaction (PCR) inhibitors, and the ability to detect biomarkers by quantitative real-time PCR and droplet digital PCR. A technique for collecting IC samples in the clinic, while preserving RNA, and a protocol for subsequent laboratory analysis of RNA were developed. RESULTS: The extracted RNA was free of PCR inhibitors and could be synthesized into complementary DNA and used for successful relative quantification of ocular surface biomarkers by quantitative real-time PCR. For gene targets present in low abundance, complementary DNA could also be used for quantification by the relatively new and emerging method of droplet digital PCR. The described method was successfully used to evaluate 3 biomarkers in a clinical trial assessing the tolerability of a proprietary eyelid therapy in 92 IC samples from a study population of 46 participants. CONCLUSIONS: IC is a recognized technique for ocular surface cell evaluation and protein biomarker quantification but is infrequently used for quantifying gene expression. The EYEPRIM device allows ease of use and impression-to-impression consistency while accurate gene expression data offers a highly specific and sensitive method of disease characterization for clinician scientists to use in diagnosis.

The Dundee Ready Education Environment Measure: A prospective comparative study of undergraduate medical students' and interns' perceptions in Oman.

OBJECTIVES: This study aimed to assess perceptions of the educational environment in Oman among medical undergraduate students and interns using the Dundee Ready Education Environment Measure (DREEM) tool. METHODS: This cross-sectional study was conducted between October 2016 and April 2017 at the Oman Medical College (OMC), Sohar, Oman. A total of 737 medical undergraduate students and interns from the OMC and College of Medicine & Health Sciences of Sultan Qaboos University in Muscat, Oman, were invited to complete the DREEM questionnaire in the form of an online survey. Mean overall scores, subscale scores and individual item scores were subsequently compared between undergraduate students and interns. RESULTS: A total of 418 undergraduate students and interns completed the survey (response rate: 56.7%). The mean overall DREEM score was 130.75 ± 12.69. While interns had higher mean DREEM scores than undergraduate students, this difference was not significant (133.00 ± 17.64 versus 128.50 ± 15.53; P = 0.326). The mean score percentages for the perceptions of learning (66.7% versus 58.3%; P = 0.028) and perceptions of teachers (75% versus 68.2%; P = 0.038) subscales were significantly higher among OMC interns compared to undergraduate students from the same college. The perceptions of the environment subscale received the lowest mean score percentages among undergraduate students and interns from both colleges. CONCLUSION: Overall, medical undergraduate students and interns viewed the educational environment in Oman in a positive light. It is possible that undergraduate students' perceptions of the educational environment may become more favourable as they progress with their medical career and become interns.

Randomized double-masked trial of eyelid cleansing treatments for blepharitis.

PURPOSE: To compare the efficacy of a dedicated eyelid cleanser and diluted baby shampoo in the management of blepharitis. METHODS: Forty-three participants with clinical blepharitis signs were enrolled in a prospective, randomized, double-masked, paired-eye trial. A dedicated eyelid cleanser (TheraTears® SteriLid®) was applied to the eyelids of one eye (randomized) and diluted baby shampoo (Johnson's® No More Tears®) to the fellow eye, twice daily for 4 weeks. Tear film parameters, ocular surface characteristics, symptomology and cytology markers were assessed at baseline and day 28. RESULTS: Baseline measurements did not differ between treatments (all p > 0.05). The eyelid cleanser was preferred over baby shampoo by the majority of participants (p < 0.001). Improvements in the tear lipid layer, inferior lid wiper epitheliopathy (LWE), cylindrical collarettes, and MMP-9 expression were limited to the dedicated eyelid cleanser (all p < 0.05), and a greater decrease in SANDE symptoms score was also observed (p = 0.04). Meibomian gland capping and MUC5AC expression worsened with baby shampoo treatment (both p < 0.05). SPEED symptoms score, superior LWE, seborrhoeic lash crusting, and trichiasis decreased significantly following application of both treatments (all p < 0.05), but did not differ between treatments (all p > 0.05). CONCLUSION: Clinical improvements in blepharitis occurred with both treatments. However, only the dedicated eyelid cleanser proved effective in reducing ocular surface inflammation, and was the preferred therapy. Long term impact of decreased goblet cell function secondary to baby shampoo treatment requires further exploration.

Implantation of Human Peripheral Corneal Spheres into Cadaveric Human Corneal Tissue.

Stem and progenitor cells isolated from human limbal tissue can be cultured in vitro as spheres. These spheres have potential for use as transplantable elements for the repopulation of corneal tissue ( Mathan et al., 2016 ). Herein we describe the detailed protocol for the implantation of human corneal spheres into cadaveric human corneal tissue. This protocol describes the procedure for sphere formation and culture, preparation of tissue for sphere implantation, corneal limbus microsurgery and sphere implantation.

Sphere-forming cells from peripheral cornea demonstrate the ability to repopulate the ocular surface.

BACKGROUND: The limbus forms the outer rim of the cornea at the corneoscleral junction and harbours a population of stem cells for corneal maintenance. Injuries to the limbus, through disease or accidents such as chemical injuries or burns, may lead to significant visual impairment due to depletion of the native stem cells of the tissue. METHODS: Sphere-forming cells were isolated from peripheral cornea for potential use as transplantable elements for limbal stem cell repopulation and limbal reconstruction. Immunocytochemistry, live cell imaging and quantitative PCR were used to characterize spheres and elucidate activity post implantation into human cadaveric corneal tissue. RESULTS: Spheres stained positively for stem cell markers ∆NP63α, ABCG2 and ABCB5 as well as the basal limbal marker and putative niche marker, notch 1. In addition, spheres also stained positively for markers of corneal cells, vimentin, keratin 3, keratocan and laminin, indicating a heterogeneous mix of stromal and epithelial-origin cells. Upon implantation into decellularized corneoscleral tissue, 3D, polarized and radially orientated cell migration with cell proliferation was observed. Cells migrated out from the spheres and repopulated the entire corneal surface over 14 days. Post-implantation analysis revealed qualitative evidence of stem, stromal and epithelial cell markers while quantitative PCR showed a quantitative reduction in keratocan and laminin expression indicative of an enhanced progenitor cell response. Proliferation, quantified by PCNA expression, significantly increased at 4 days subsequently followed by a decrease at day 7 post implantation. CONCLUSION: These observations suggest great promise for the potential of peripheral corneal spheres as transplantable units for corneal repair, targeting ocular surface regeneration and stem cell repopulation.

Limbal stem cells: Central concepts of corneal epithelial homeostasis.

A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent studies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface.

Efficacy of a novel light-activated antimicrobial coating for disinfecting hospital surfaces.

Silicone polymers containing the light-activated antimicrobial agent methylene blue with or without gold nanoparticles were evaluated for their ability to reduce the microbial load on surfaces in a clinical environment. When irradiated with white light, polymers containing nanogold were more effective in this respect than those containing only methylene blue.

Counterstaining improves visualization of the myenteric plexus in immunolabelled whole-mount preparations.

Immunocytochemistry has emerged as a powerful research tool in neurobiology. One of the widely used methods is an indirect fluorescence technique that uses FITC- conjugated IgG to visualise protein expression within tissues, but a major drawback of this technique is the high background fluorescence due to non-specific antibody binding. Gut innervation is complex and best visualized in three-dimensions in whole mount preparations. We describe a simple and easy to use counterstaining procedure in conjunction with an indirect immunofluorescence technique in gut whole mount preparations that largely eliminates background fluorescence and creates a contrasting background against the bright antigen-antibody complexes. Furthermore, this technique allows the detailed qualitative and quantitative study of myenteric plexuses in whole-mount preparations.

Helicobacter pylori outer membrane vesicles modulate proliferation and interleukin-8 production by gastric epithelial cells.

Helicobacter pylori infection, which is always associated with gastritis, can progress to ulceration or malignancy. The diversity in clinical outcomes is partly attributed to the expression of virulence factors and adhesins by H. pylori. However, H. pylori may not have to adhere to the epithelium to cause gastritis. We hypothesize that outer membrane vesicles (OMV), which are constantly shed from the surface of H. pylori, play a role as independent activators of host cell responses. In this study, we found that low doses of OMV from cag PAI+ toxigenic and cag PAI- nontoxigenic strains increased proliferation of AGS gastric epithelial cells. At higher doses, we detected growth arrest, increased toxicity, and interleukin-8 (IL-8) production. The only strain differences detected were vacuolation with the toxigenic strain and higher levels of IL-8 production with OMV from the cag PAI- nontoxigenic strain. In summary, we suggest that constitutively shed OMV play a role in promoting the low-grade gastritis associated with H. pylori infection.

The action of bismuth against Helicobacter pylori mimics but is not caused by intracellular iron deprivation.

Helicobacter pylori is highly susceptible to bismuth, a heavy metal with antimicrobial activity linked to its effect on bacterial iron uptake. Three strains of H. pylori were analyzed for indicators of iron limitation following exposure to the MIC of colloidal bismuth subcitrate (MIC(CBS)). Similar morphologic and outer membrane changes were observed following growth in iron-limiting medium and at the MIC(CBS) that inhibited the growth of all three strains. These changes, which were also observed for iron-limited bacteria, were alleviated by the addition of iron to the cultures. H. pylori ATP levels, reduced in iron-limiting medium, were below the limits of detection in two of the three strains following exposure to bismuth. The addition of iron partially restored bacterial ATP levels in these two strains, although not to normal concentrations. In contrast, exposure of the same strains to the MIC(CBS) failed to deplete intracellular levels of iron, which were significantly reduced by culturing in iron-limiting medium. Thus, the antimicrobial effect of bismuth and of iron limitation on H. pylori may be similar. However, the respective mechanisms of intracellular action would appear to be mediated by different pathways within the cell.