Eradication treatment for Pseudomonas aeruginosa infection in adults with bronchiectasis: a systematic review and meta-analysis.

INTRODUCTION: Pseudomonas aeruginosa is the most commonly isolated pathogen in bronchiectasis and is associated with worse outcomes. Eradication treatment is recommended by guidelines, but the evidence base is limited. The expected success rate of eradication in clinical practice is not known. METHODS: We conducted a systematic review and meta-analysis according to Meta-Analysis of Observational Studies in Epidemiology guidelines. PubMed, Embase, the Cochrane Database of Systematic Reviews and Clinicaltrials.gov were searched for studies investigating P. aeruginosa eradication treatment using antibiotics (systemic or inhaled) in patients with bronchiectasis. The primary outcome was the percentage of patients negative for P. aeruginosa at 12 months after eradication treatment. Cystic fibrosis was excluded. RESULTS: Six observational studies including 289 patients were included in the meta-analysis. Our meta-analysis found a 12-month P. aeruginosa eradication rate of 40% (95% CI 34-45%; p<0.00001), with no significant heterogeneity (I2=0%). Combined systemic and inhaled antibiotic treatment was associated with a higher eradication rate (48%, 95% CI 41-55%) than systemic antibiotics alone (27%, 13-45%). CONCLUSION: Eradication treatment in bronchiectasis results in eradication of P. aeruginosa from sputum in ∼40% of cases at 12 months. Combined systemic and inhaled antibiotics achieve higher eradication rates than systemic antibiotics alone.

How to Manage Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is a pathogen frequently encountered in healthcare-associated infections and immunocompromised patients. In bacteremia, this pathogen is associated with higher mortality than other Gram-negative pathogens. This increase in mortality was also found globally for multi-resistant compared to susceptible strains. Several factors have been associated with the development of resistance: previous ICU stay, use of carbapenems, and comorbidities were identified in multivariate analysis. In the therapeutic choice, previous antibiotic treatment remains the strongest driver suggesting a potential resistant strain. These risk factors will decide whether multi-resistant strains must be considered in the empiric coverage. For susceptible strains, a single agent can be used, ß-lactams are usually the first choice. Associations do not provide any advantage on mortality. Optimization of pharmacokinetic/pharmacodynamic parameters, such as prolonged infusion (for time-dependent antibiotics), increased dosage (for concentration-dependent antibiotics), and therapeutic drug monitoring, also influences the outcome. The increasing number of resistant strains led the clinician to use either recently approved new molecules but also associations. For multi-resistant strains, new molecules such as ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol have shown an adequate activity against P. aeruginosa. Older molecules like colistin and fosfomycin are also used in this indication. The complexity of the resistance and consequences on a larger scale of antibiotic prescription will probably lead to more individualized prescriptions.

Zinc Exposure Promotes Commensal-to-Pathogen Transition in Pseudomonas aeruginosa Leading to Mucosal Inflammation and Illness in Mice.

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) is associated gastrointestinal (GI) inflammation and illness; however, factors motivating commensal-to-pathogen transition are unclear. Excessive zinc intake from supplements is common in humans. Due to the fact that zinc exposure enhances P. aeruginosa colonization in vitro, we hypothesized zinc exposure broadly activates virulence mechanisms, leading to inflammation and illness. P. aeruginosa was treated with excess zinc and growth, expression and secretion of key virulence factors, and biofilm production were determined. Effects on invasion, barrier function, and cytotoxicity were evaluated in Caco-2 cells co-cultured with P. aeruginosa pre-treated with zinc. Effects on colonization, mucosal pathology, inflammation, and illness were evaluated in mice infected with P. aeruginosa pre-treated with zinc. We found the expression and secretion of key virulence factors involved in quorum sensing (QS), motility (type IV pili, flagella), biosurfactants (rhamnolipids), toxins (exotoxin A), zinc homeostasis (CzcR), and biofilm production, were all significantly increased. Zinc exposure significantly increased P. aeruginosa invasion, permeability and cytotoxicity in Caco-2 cells, and enhanced colonization, inflammation, mucosal damage, and illness in mice. Excess zinc exposure has broad effects on key virulence mechanisms promoting commensal-to-pathogen transition of P. aeruginosa and illness in mice, suggesting excess zinc intake may have adverse effects on GI health in humans.

Evaluation of Antibiotic Resistance and Virulence Genes among Clinical Isolates of Pseudomonas aeruginosa from Cancer Patients.

OBJECTIVES: The objectives of this study were to evaluate P. Aeruginosa isolates from cancer patients for the phenotypic pattern of antibiotic resistance and to detect the gene responsible for virulence as well as antibiotic resistance. METHODS: A total of 227 P. aeruginosa isolates were studied and 11 antibiotics were applied for susceptibility testing. PCR detection of the genes BIC, TEM, IMP, SPM, AIM, KPC, NDM, GIM, VIM, OXA, toxA and oprI was done. Finally, the carbapenem resistant isolates were tested for phenotypic identification of carbapenemase enzyme by Modified Hodge test. RESULTS: The results showed that the isolates were resistant to imipenem (95%), cefipime (93%), meropenem (90%), polymixin B (71%), gentamicin (65%), ciprofloxacin (48%), ceftazidime (40%), levofloxacin (39%), amikacin (32%), tobramycin (28%) and tazobactum (24%). The PCR detection of the carbapenem resistant genes showed 51% isolates were positive for IMP, GIM and VIM, 38% for AIM and SPM, 30% for BIC, 20% for TEM and NDM, 17% for KPC and 15% for OXA. However, toxA and oprI genes were not detected. 154 carbapenem resistant isolates were found positive phenotypically for carbapenemase enzyme identification by Modified Hodge test. CONCLUSION: The co-existence of multiple drug-resistant bodies and virulent genes has important implications for the treatment of patients. This study provides information about treating drug-resistant P. Aeruginosa and the relationship of virulent genes with phenotypic resistance patterns.
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Meropenem to Children With Febrile Neutropenia Induces Monoresistant Pseudomonas aeruginosa.

Antimicrobial resistance in Pseudomonas aeruginosa is a threat to children with cancer. We explored the association between P. aeruginosa resistance and previous antibiotic exposure. All children with cancer and P. aeruginosa bacteremia in 2007 to 2016 in Denmark, a country with an overall resistance rate of ∼3%, were included. Twenty percent (10/49) of isolates from children previously exposed to meropenem were meropenem nonsusceptible. The only significant risk factor of meropenem nonsusceptibility was previous meropenem therapy (P=0.03). On the basis of these results, we suggest that meropenem should be reserved as a last resort for children with febrile neutropenia in countries with low antimicrobial resistance.

Synergistic Activity of Berberine with Azithromycin against Pseudomonas Aeruginosa Isolated from Patients with Cystic Fibrosis of Lung In Vitro and In Vivo.

BACKGROUND/AIMS: Pseudomonas aeruginosa (PA) is one of the major opportunistic pathogens which can cause chronic lung infection of cystic fibrosis (CF). The formation of PA biofilm promotes CF development and restricts the antimicrobial efficacies of current antibiotics. METHODS: The antimicrobial effects of azithromycin (AZM) and berberine (BER) alone and in combination were evaluated using microdilution method, checkerboard assay, time-kill test, qRT-PCR analysis and absorption method. The treatments of AZM and/or BER were further evaluated in an animal lung infection model via observing survival rate, bacterial burden and histopathology of lung, the levels of pro-/anti-inflammatory cytokines. RESULTS: AZM-BER were demonstrated to be synergistic against ten clinical PA isolates as well as the standard reference PA ATCC27853, in which PA03 was the most susceptible isolate to AZM-BER with FICI of 0.13 and chosen for subsequent experiments. The synergism of AZM-BER was further confirmed against PA03 in time-kill test and scanning electron microscope (SEM) at their concentrations showing synergism. In PA03, we found that AZM-BER could significantly attenuate productions of a series of virulence factors including alginate, LasA protease, LasB protease, pyoverdin, pyocyanin, chitinase as well as extracellular DNA, and remarkably inhibit the levels of quorum sensing (QS) molecules and the expressions of lasI, lasR, rhlI, rhlR at 1/2×MIC, 1×MIC and 2×MIC. In the infection model, the mice survival were increased markedly, the inflammations of infected lungs were improved greatly along with reduced IL-6, IL-8 and ascended IL-10 at 0.8 mg/kg of AZM combined with 3.2 mg/kg of BER. CONCLUSION: BER might be a promising synergist to enhance the antimicrobial activity of AZM in vitro and in vivo.

Delayed diagnosis of central skull-base osteomyelitis with abscess: case report and learning points.

Central skull-base osteomyelitis (CSBO) is a rare life-threatening infection, usually resulting from medial spread of necrotising otitis externa. Here, we describe a case with no identifiable source of infection, causing a delay in diagnosis. An 80-year-old man with Crohn's disease treated with mesalazine presented with collapse and tonic-clonic seizure. Computed tomography and magnetic resonance imaging showed a nasopharyngeal mass that was initially thought to be a neoplasm. Awaiting formal biopsy, he represented with collapse and repeat imaging showed features of abscess formation. Review of previous scans revealed skull-base erosion and the diagnosis was revised to skull-base osteomyelitis. This is the first reported case of CSBO associated with mesalazine use, an aminosalicylate used in Crohn's disease. It is only the second reported case with abscess formation. We discuss the learning points in making a timely diagnosis and examine the potential association of factors such as mesalazine use and abscess formation in this case.

Acute exposure to silica nanoparticles enhances mortality and increases lung permeability in a mouse model of Pseudomonas aeruginosa pneumonia.

BACKGROUND: The lung epithelium constitutes the first barrier against invading pathogens and also a major surface potentially exposed to nanoparticles. In order to ensure and preserve lung epithelial barrier function, the alveolar compartment possesses local defence mechanisms that are able to control bacterial infection. For instance, alveolar macrophages are professional phagocytic cells that engulf bacteria and environmental contaminants (including nanoparticles) and secrete pro-inflammatory cytokines to effectively eliminate the invading bacteria/contaminants. The consequences of nanoparticle exposure in the context of lung infection have not been studied in detail. Previous reports have shown that sequential lung exposure to nanoparticles and bacteria may impair bacterial clearance resulting in increased lung bacterial loads, associated with a reduction in the phagocytic capacity of alveolar macrophages. RESULTS: Here we have studied the consequences of SiO2 nanoparticle exposure on Pseudomonas aeruginosa clearance, Pseudomonas aeruginosa-induced inflammation and lung injury in a mouse model of acute pneumonia. We observed that pre-exposure to SiO2 nanoparticles increased mice susceptibility to lethal pneumonia but did not modify lung clearance of a bioluminescent Pseudomonas aeruginosa strain. Furthermore, internalisation of SiO2 nanoparticles by primary alveolar macrophages did not reduce the capacity of the cells to clear Pseudomonas aeruginosa. In our murine model, SiO2 nanoparticle pre-exposure preferentially enhanced Pseudomonas aeruginosa-induced lung permeability (the latter assessed by the measurement of alveolar albumin and IgM concentrations) rather than contributing to Pseudomonas aeruginosa-induced lung inflammation (as measured by leukocyte recruitment and cytokine concentration in the alveolar compartment). CONCLUSIONS: We show that pre-exposure to SiO2 nanoparticles increases mice susceptibility to lethal pneumonia but independently of macrophage phagocytic function. The deleterious effects of SiO2 nanoparticle exposure during Pseudomonas aeruginosa-induced pneumonia are related to alterations of the alveolar-capillary barrier rather than to modulation of the inflammatory responses.

HIF-1α is essential for effective PMN bacterial killing, antimicrobial peptide production and apoptosis in Pseudomonas aeruginosa keratitis.

Hypoxia-inducible factor (HIF)-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals) mice after ocular infection with Pseudomonas (P.) aeruginosa. Furthermore, the current studies focused on the neutrophil (PMN), the predominant cell infiltrate in keratitis. Using both siRNA and an antagonist (17-DMAG), the role of HIF-1α was assessed in P. aeruginosa-infected BALB/c mice. Clinical score and slit lamp photography indicated HIF-1α inhibition exacerbated disease and corneal destruction. Real time RT-PCR, immunohistochemistry, ELISA, Greiss and MPO assays, bacterial load, intracellular killing, phagocytosis and apoptosis assays further tested the regulatory role of HIF-1α. Despite increased pro-inflammatory cytokine expression and increased MPO levels after knocking down HIF-1α expression, in vivo studies revealed a decrease in NO production and higher bacterial load. In vitro studies using PMN provided evidence that although inhibition of HIF-1α did not affect phagocytosis, both bacterial killing and apoptosis were significantly affected, as was production of antimicrobial peptides. Overall, data provide evidence that inhibition of HIF-1α converts a normally resistant disease response to susceptible (corneal thinning and perforation) after induction of bacterial keratitis. Although this inhibition does not appear to affect PMN transmigration or phagocytosis, both in vivo and in vitro approaches indicate that the transcriptional factor is essential for effective bacterial killing, apoptosis and antimicrobial peptide production.

Surfactant protein D contributes to ocular defense against Pseudomonas aeruginosa in a murine model of dry eye disease.

Dry eye disease can cause ocular surface inflammation that disrupts the corneal epithelial barrier. While dry eye patients are known to have an increased risk of corneal infection, it is not known whether there is a direct causal relationship between these two conditions. Here, we tested the hypothesis that experimentally-induced dry eye (EDE) increases susceptibility to corneal infection using a mouse model. In doing so, we also examined the role of surfactant protein D (SP-D), which we have previously shown is involved in corneal defense against infection. Scopolamine injections and fan-driven air were used to cause EDE in C57BL/6 or Black Swiss mice (wild-type and SP-D gene-knockout). Controls received PBS injections and were housed normally. After 5 or 10 days, otherwise uninjured corneas were inoculated with 10(9) cfu of Pseudomonas aeruginosa strain PAO1. Anesthesia was maintained for 3 h post-inoculation. Viable bacteria were quantified in ocular surface washes and corneal homogenates 6 h post-inoculation. SP-D was measured by Western immunoblot, and corneal pathology assessed from 6 h to 4 days. EDE mice showed reduced tear volumes after 5 and 10 days (each by ∼75%, p<0.001) and showed fluorescein staining (i.e. epithelial disruption). Surprisingly, there was no significant difference in corneal pathology between EDE mice and controls (∼10-14% incidence). Before bacterial inoculation, EDE mice showed elevated SP-D in ocular washes. After inoculation, fewer bacteria were recovered from ocular washes of EDE mice (<2% of controls, p = 0.0004). Furthermore, SP-D knockout mice showed a significant increase in P. aeruginosa corneal colonization under EDE conditions. Taken together, these data suggest that SP-D contributes to corneal defense against P. aeruginosa colonization and infection in EDE despite the loss of barrier function to fluorescein.

Estrogen aggravates inflammation in Pseudomonas aeruginosa pneumonia in cystic fibrosis mice.

BACKGROUND: Among patients with cystic fibrosis (CF), females have worse pulmonary function and survival than males, primarily due to chronic lung inflammation and infection with Pseudomonas aeruginosa (P. aeruginosa). A role for gender hormones in the causation of the CF "gender gap" has been proposed. The female gender hormone 17ß-estradiol (E2) plays a complex immunomodulatory role in humans and in animal models of disease, suppressing inflammation in some situations while enhancing it in others. Helper T-cells were long thought to belong exclusively to either T helper type 1 (Th1) or type 2 (Th2) lineages. However, a distinct lineage named Th17 is now recognized that is induced by interleukin (IL)-23 to produce IL-17 and other pro-inflammatory Th17 effector molecules. Recent evidence suggests a central role for the IL-23/IL-17 pathway in the pathogenesis of CF lung inflammation. We used a mouse model to test the hypothesis that E2 aggravates the CF lung inflammation that occurs in response to airway infection with P. aeruginosa by a Th17-mediated mechanism. RESULTS: Exogenous E2 caused adult male CF mice with pneumonia due to a mucoid CF clinical isolate, the P. aeruginosa strain PA508 (PA508), to develop more severe manifestations of inflammation in both lung tissue and in bronchial alveolar lavage (BAL) fluid, with increased total white blood cell counts and differential and absolute cell counts of polymorphonuclear leukocytes (neutrophils). Inflammatory infiltrates and mucin production were increased on histology. Increased lung tissue mRNA levels for IL-23 and IL-17 were accompanied by elevated protein levels of Th17-associated pro-inflammatory mediators in BAL fluid. The burden of PA508 bacteria was increased in lung tissue homogenate and in BAL fluid, and there was a virtual elimination in lung tissue of mRNA for lactoferrin, an antimicrobial peptide active against P. aeruginosa in vitro. CONCLUSIONS: Our data show that E2 increases the severity of PA508 pneumonia in adult CF male mice, and suggest two potential mechanisms: enhancement of Th17-regulated inflammation and suppression of innate antibacterial defences. Although this animal model does not recapitulate all aspects of human CF lung disease, our present findings argue for further investigation of the effects of E2 on inflammation and infection with P. aeruginosa in the CF lung.

[An unusual presentation of Fournier's gangrene. Is there a relationship with NSAID use? Pediatric case report]. / Gangrena de Fournier en asociación con el uso de anti-inflamatorios no esteroideos. reporte de un caso pediátrico.

Fournier's gangrene is a rare but life-threatening disease characterized by necrotizing fasciitis of the perineal area. It mostly affects adult patients with an immunosuppressant factor and there are only a few cases reported in children. Use of nonsteroidal anti-inflammatory drugs has been associated with necrotizing fasciitis probably because of suppressing host immunity during infection. We describe a case of a six month old infant without immunosuppressant factors, with Founier's gangrene probably associated with nonsteroidal anti-inflammatory intake.

Gangrena de Fournier en asociación con el uso de anti-inflamatorios no esteroideos: Reporte de un caso pediátrico / An unusual presentation of Fournier's gangrene: ¿Is there a relationship with NSAID use? Pediatric case report

La gangrena de Fournier es una enfermedad amenazante para la vida, que se caracteriza por fascitis necrosante del área perineal. Afecta con mayor frecuencia a pacientes adultos con inmunosupresión y son pocos los casos reportados en la población pediátrica. El uso de fármacos anti-inflamatorios no esteroideos (AINEs) se ha asociado con fascitis necrosante, probablemente por supresión de la inmunidad durante la infección. Describimos el caso de un paciente con 6 meses de edad sin factores inmu-nosupresores, con gangrena de Fournier, probablemente asociada a la ingesta de AINEs.

Founier's gangrene is a rare but life-threatening disease characterized by necrotizing fasciitis of the perineal area. It mostly affects adult patients with an immunosuppressant factor and there are only a few cases reported in children. Use of nonsteroidal anti-inflammatory drugs has been associated with necrotizing fasciitis probably because of suppressing host immunity during infection. We describe a case of a six month old infant without im-munosuppressant factors, with Founier´s gangrene probably associated with nonsteroidal anti-inflammatory intake.

Severe necrotizing soft-tissue infections and nonsteroidal anti-inflammatory drugs.

BACKGROUND: Severe necrotizing soft-tissue infection (NSTI) is a rare but potentially life-threatening condition if not recognized and treated early. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been implicated as a contributing factor, but their role remains debated. AIMS: The aim of our study was to investigate the potential relationship between cases of NSTI recorded in the French Pharmacovigilance system and exposure to NSAIDs. METHODS: Cases of NSTI and randomly selected matched noncase controls (without skin disease) were identified in the database of the Spontaneous Reporting System in France for the period 2000-2004. Exposure to NSAIDs and other factors were investigated using conditional logistic regression. RESULTS: We found 38 cases of NSTI in 2000-04: 12 infants (0-23 months), 16 children (2-15 years) and 10 adults (>15 years), and we selected 228 controls. The median age of the sample was 4 years. Of the 38 cases, 25 were exposed to ibuprofen and 24 presented with varicella. The adjusted odds ratio for exposure to NSAIDs was 31.38 (95% CI 6.40-153.84), and 17.55 (95% CI 3.47-88.65) for viral infection. Other predisposing factors (diabetes, immunosuppression, injecting drugs) were not found to be associated, although this may have been due to the very small number of cases of NSTI/necrotizing fasciitis in adults reported in the database. CONCLUSION: Despite the limitations related to a spontaneous reporting system, this study indicates a strong association between NSAID use and NSTI. Although it was not possible to conclude if NSAIDs increase the risk of necrotizing complications in all patients, their use may mask the symptoms and delay diagnosis.

TLR4 signaling is essential for survival in acute lung injury induced by virulent Pseudomonas aeruginosa secreting type III secretory toxins.

BACKGROUND: The relative contributions of the cytotoxic phenotype of P. aeruginosa expressing type III secretory toxins and an immunocompromised condition lacking normal Toll-like receptor 4 (TLR4) signaling in the pathogenesis of acute lung injury and sepsis were evaluated in a mouse model for Pseudomonas aeruginosa pneumonia. By using lipopolysaccharide-resistant C3H/HeJ mice missing normal TLR4 signaling due to a mutation on the tlr4 gene, we evaluated how TLR4 signaling modulates the pneumonia caused by cytotoxic P. aeruginosa expressing type III secretory toxins. METHODS: We infected C3H/HeJ or C3H/FeJ mice with three different doses of either a cytotoxic P. aeruginosa strain (wild type PA103) or its non-cytotoxic isogenic mutant missing the type III secretory toxins (PA103DeltaUT). Survival of the infected mice was evaluated, and the severity of acute lung injury quantified by measuring alveolar epithelial permeability as an index of acute epithelial injury and the water to dry weight ratios of lung homogenates as an index of lung edema. Bacteriological analysis and cytokine assays were performed in the infected mice. RESULTS: Development of acute lung injury and sepsis was observed in all mouse strains when the cytotoxic P. aeruginosa strain but not the non-cytotoxic strain was instilled in the airspaces of the mice. Only C3H/HeJ mice had severe bacteremia and high mortality when a low dose of the cytotoxic P. aeruginosa strain was instilled in their lungs. CONCLUSION: The cytotoxic phenotype of P. aeruginosa is the critical factor causing acute lung injury and sepsis in infected hosts. When the P. aeruginosa is a cytotoxic strain, the TLR4 signaling system is essential to clear the bacteria to prevent lethal lung injury and bacteremia.

[An auto-iatrogenic disease]. / Ein auto-iatrogenes Leiden.

A 55-year-old practitioner from an island in the northern sea felt an increasing hypersensitivity of his entire body to various ambient and nutritional allergens and toxics. He started to treat himself with increasing doses of glucocorticoids and moved to a southern climate in Lanzarote and later on to the Swiss mountains in the grisons. On admission to our hospital in December he was in a disastrous psychotic condition, trying to cool down his body by laying naked on his bed at ambient temperatures around the freezing point. He had consumed on average 250 mg prednisone daily over weeks. As we found out later his personal assistant travelling with him was giving him glucocorticoids through the infusion during his hospital stay. He developed a necrotizing septic phlebitis at the infusion site followed by a Pseudomonas aeruginosa sepsis with fatal multiorgan failure. This case illustrates the dangers of self-treatment by doctors and the difficulties in treating a physician.

Cancrum oris (noma) in a patient with acute lymphoblastic leukaemia. A complication of chemotherapy induced neutropenia.

Cancrum oris (noma) has been most commonly described in malnourished debilitated children with poor oral hygiene following systemic childhood infections such as measles, pertussis or scarlet fever. We describe a patient who developed this condition during a period of profound neutropenia following cytotoxic chemotherapy for acute lymphoblastic leukaemia.

Pseudomonas aeruginosa protease IV produces corneal damage and contributes to bacterial virulence.

PURPOSE: A Pseudomonas mutant deficient in protease IV has significantly reduced virulence in experimental keratitis. In the present study, the corneal toxicity of purified protease IV and its ability to augment the virulence of protease-IV-deficient bacteria were analyzed. METHODS: The toxicity of purified protease IV was determined by intrastromally injecting the exoenzyme (20-200 ng) into the cornea. The effects of protease IV on the corneal virulence of the protease-IV-deficient strain, PA103-29::Tn9, were determined by injecting eyes with 1000 CFU of log phase bacteria plus either 200 ng active purified protease IV or 200 ng heat-inactivated protease IV. Changes in ocular disease, determined by slit-lamp examination, were measured at 3, 16, 22, and 27 hours after infection. Colony-forming units per cornea were quantified at 27 hours after infection. RESULTS: Purified protease IV at doses from 50 to 200 ng induced epithelial defects within 3 hours of injection. Injection of 20 ng active protease IV or heat-inactivated protease IV (200 ng) had no effect on ocular tissue. Corneal virulence of the protease-IV-deficient strain was augmented by intrastromal injection with purified protease IV but not with heat-inactivated protease IV (P < or = 0.0001). Neither active nor heat-inactivated protease IV altered the growth of bacteria in the cornea (6 log units; P = 0.81). CONCLUSIONS: The important role of protease IV in corneal virulence was demonstrated by direct toxicity and by its ability to significantly augment the virulence of protease-IV-deficient Pseudomonas.