Proteinases of Pseudomonas aeruginosa evoke mucin release by tracheal epithelium.

We have determined the potential of exoproducts from pathogenic bacteria to stimulate the release of high molecular weight mucins from goblet cells of airway epithelium in a rabbit tracheal explant system. Culture supernatants from proteolytic strains of Pseudomonas aeruginosa and Serratia marcescens, but not supernatants from a number of non-proteolytic strains, released mucins from goblet cells. Highly purified elastase and alkaline proteinase from P. aeruginosa stimulated goblet cell mucin release in a dose-dependent fashion. Lipopolysaccharide, exotoxin A, and alginate of P. aeruginosa did not possess mucin release properties. Proteolytic activity was required for mucin release by P. aeruginosa elastase, but such release in goblet cells was not mediated by cyclic AMP. Morphologic studies suggested rapid release of mucins from goblet cells was response to elastase by a process resembling apocrine secretion. Several nonbacterial proteinases mimicked the effect of Pseudomonas proteases. These studies provide support for the hypothesis that bacterial and other play a role in the pathogenesis of mucus hypersecretion in acute and chronic lung infections.

Ceftazidime therapy in patients with cystic fibrosis and multiply-drug-resistant pseudomonas.

The in vitro activity of ceftazidime against Pseudomonas aeruginosa and P. cepacia isolates from patients with cystic fibrosis was compared with that of other antipseudomonal drugs. Ceftazidime was as potent as imipenem against P. aeruginosa and the only drug effective against P. cepacia. An evaluation of the elimination kinetics of ceftazidime in 20 cystic fibrosis patients revealed an elimination half-life of 1.76 hours, an apparent distribution volume of 0.27 liters/kg, and a serum clearance rate of 133.9 ml/minute/1.73m2. Urinary recovery of ceftazidime was 87 percent within the first 24 hours after administration of the drug, with 65 percent recovered in the first two-hour fraction. Probenecid administration had no effect on the elimination kinetics of ceftazidime. Forty-three patients who had either shown no response to conventional therapy or had sputum Pseudomonas isolates that were susceptible only to ceftazidime received 75 courses of therapy. In 67 percent of these patients, the clinical response, when evaluated using an objective clinical efficacy scoring system, was considered favorable. Clinical failures were not associated with the development of drug resistance. Thus, ceftazidime can be recommended for the treatment of acute pulmonary exacerbations in patients with cystic fibrosis.

Colonization of the respiratory tract with Pseudomonas cepacia in cystic fibrosis. Risk factors and outcomes.

Between 1981 and 1983, some 85 patients with cystic fibrosis at Rainbow Babies and Childrens Hospital, Cleveland, developed colonization or infection of the respiratory tract with Pseudomonas cepacia. Twenty-nine (34 percent) of the colonized patients died; four were female patients with fulminant bacteremia with P cepacia prior to death. Case-control studies showed that increasing severity of underlying cystic fibrosis, increasing age, having a sibling with cystic fibrosis who was colonized with P cepacia, and previous hospitalizations were associated with increased risk of colonization. In patients with mild cystic fibrosis, no differences in clinical outcome were seen during the period of study; however, patients colonized with P cepacia who had moderate or advanced cystic fibrosis were hospitalized longer and died sooner after colonization, compared with control subjects with similar severity of cystic fibrosis. The excess mortality associated with such colonization varied in magnitude and trend according to the patient's sex and severity of underlying cystic fibrosis, reflecting the combined influence of colonization with P cepacia, sex, and severity of cystic fibrosis on the mortality of the patients. The source and mode of transmission of P cepacia were not determined, but the data suggest a possible nosocomial source. The results of this investigation showed that colonization with P cepacia most often affected patients with moderate or advanced cystic fibrosis and was associated with an adverse clinical outcome in these patients.

Occurrence and antimicrobial susceptibility of gram-negative nonfermentative bacilli in cystic fibrosis patients.

Isolation of nonfermentative gram-negative bacilli (other than Pseudomonas aeruginosa) from respiratory tract cultures of cystic fibrosis (CF) patients has increased in recent years. Species recovered include Pseudomonas cepacia, P. maltophilia, P. fluorescens/putida, P. alcaligenes, P. pseudoalcaligenes, P. stutzeri, Acinetobacter spp., Achromobacter xylosoxidans, Flavobacterium spp., and CDC groups IVe and Ve. Although colonization with most of these organisms is sporadic, P. cepacia (and to a lesser extent, P. maltophilia) is usually isolated consistently, and can be associated with significant clinical deterioration. Occurrence of P. cepacia in CF respiratory tract cultures obtained close to the time of death rose nearly ten-fold from 1979 to 1982. Strains representing all nonfermentative gram-negative species encountered were assayed for susceptibility to 17 newer antimicrobial agents. Ceftazidime, n-formimidoyl thienamycin, and aztreonam were most active; cefsulodin, ceforanide, and ceftriaxone were not active against these isolates.

Therapeutic evaluation of piperacillin for acute pulmonary exacerbations in cystic fibrosis.

The efficacy and pharmacokinetics of piperacillin monotherapy were studied in 46 patients with cystic fibrosis. Two patients were dropped from the study within 24 hr of enrollment because of drug-associated nausea and vomiting. Initially fourteen older patients (greater than 12 years) receiving piperacillin 450 mg/kg/day underwent a preliminary evaluation. Based on the results, 30 younger patients (less than or equal to 12 years) randomized in a double-blind fashion received either 600 or 900 mg/kg/day of piperacillin in six divided doses. Pharmacokinetic parameter estimates for t1/2 Vdss, and Cl were similar for first dose and steady-state evaluations. In 27 patients, approximately 43% of the administered dose was recovered in the urine after 4 hr. Piperacillin CiR averaged 49% of the total Cl. No difference in overall clinical efficacy could be identified between 600 and 900 mg/kg/day of piperacillin using two different objective scoring systems. Although a reduction in sputum Pseudomonas colony counts was greater following the 900 mg/kg/day regimen, this appeared to be independent of clinical effect. In 14 patients (32%), a distinct adverse serum-sicknesslike reaction was observed. The incidence of this reaction appeared to increase as the dose of piperacillin increased. All signs and symptoms of this reaction resolved within 36 hr of discontinuing piperacillin administration but recurred immediately on rechallenge in four patients. All patients with the adverse reaction were subsequently treated with beta-lactam antibodies without ill effect. Overall, clinical improvement appeared to be independent of the piperacillin dose. Our data support the use of total daily piperacillin dosages not exceeding 600 mg/kg.

A new colorimetric nucleic acid hybridization assay for Listeria in foods.

Non-isotopic colorimetric detection has been applied in a rapid nucleic acid dipstick hybridization assay for detection of Listeria spp. in food and environmental samples. The assay takes approximately 2.5-3 h following a two day broth and plate enrichment. Hybridization occurs between fluorescein labeled detector probes, poly(deoxyadenosine)-tailed capture probes, and Listeria-specific regions of 16 S ribosomal RNA. These target:probe complexes are captured on poly(deoxythymidine) coated plastic dipsticks. Detection is based on binding of horseradish peroxidase conjugated anti-fluorescein antibody to the hybridization complex, and enzyme-mediated color development. The colorimetric endpoint is read on a photometer at 450 nm. In these initial studies, 306 inoculated dairy, meat and seafood samples, and 200 environmental samples were tested. When compared with total culture results the hybridization assay had unconfirmed positive and false-negative rates of approximately 1.4-2.9% and 0.8-4.7%, respectively.

An in-hospital evaluation of the sonic mist ultrasonic room humidifier.

It is generally recognized that nebulizers can be a source of nosocomial infection. 'Cold mist' room humidifiers are a particular problem because they are difficult to sterilize. We evaluated a new device, the Sonic Mist ultrasonic room humidifier, to determine how quickly it became contaminated during continuous use by a population of cystic fibrosis patients. In addition, the study was designed to test the effectiveness of placing a bacterial filter on the air inlet of the humidifier. We found that the entire humidifier could withstand repeated gas sterilization. Data obtained from 18 humidifiers involving cystic fibrosis patients indicate that the earliest humidifier contamination occurred after 5 days of continuous use. Although all patients had large numbers of gram-negative bacilli as predominant sputum flora, only 7 episodes of contamination were found during 34 humidifier-use periods. Three units equipped with filters became contaminated (5-7 days) and four unfiltered units became contaminated (6-11 days), indicating that the use of an inlet filter made no apparent difference. The organisms recovered from contaminated units were not found as sputum flora and would not generally be considered of clinical significance in cystic fibrosis sputum cultures. Probable sources of the organisms were room dust and hand contamination. A further test of the inlet filter was performed by exposing filtered and unfiltered units to mist from an intentionally contaminated humidifier. Again, the contamination rate was low and the filter apparently made no difference. These results indicate that the Sonic Mist humidifier may be appropriate for hospital use if adequate sterilization and contamination-monitoring practices are followed.

Identification of Salmonella enteritidis from experimentally infected hens using a colorimetric DNA hybridization method.

Identification of Salmonella enteritidis from cloacally challenged commercial laying hens was studied by comparing bacterial isolations using conventional methods with detection by the use of the GENE-TRAK colorimetric DNA probe assay. More positive test results were obtained using the latter on days 14, 28, and 42 postchallenge, but the difference between the two methods was not statistically significant. Over the duration of the experiment, positive cloacal samples were statistically more frequent from a commercial strain of white leghorn hens when compared with a commercial brown egg-producing strain (28/60 vs. 9/57; chi-square 1 df = 12.9, P < 0.001). Eggs having various shell defects were produced by the infected hens only after Salmonella challenge. These defects included, in order of frequency, elongated shape, thin shells, off-white color (tints), small size, wrinkles, and pimples. No Salmonella could be recovered from 193 defective eggs, nor were positive isolates made from additional tests performed on 50 normal eggs. Proteus sp. was isolated from 10 eggs, however. Our observations demonstrate that the GENE-TRAK colorimetric method is comparable with conventional bacteriology for the identification of Salmonella in cloacal samples taken from laying hens. Moreover, the two methods demonstrate the existence of breed differences in susceptibility to S. enteritidis challenge.

Some visual effects caused by the beverage kava.

Kava is a drink produced from the plant Piper methysticum and used as a social and ceremonial beverage on many South Pacific islands. Visual functions were measured in one subject following the taking of this drink. A reduced near point of accommodation and convergence, an increase in pupil diameter and disturbance to the oculomotor balance were noted. No changes were recorded in visual or stereoacuity or in ocular refractive error.

Isolation of Listeria: a review of procedures and future prospects.

Recent documented foodborne outbreaks of listeriosis have underscored the need for improved isolation and identification procedures for Listeria. This review considers the development of selective enrichment media, various approaches to improving efficiency of isolation, and efforts to shorten enrichment periods. The application of simplified rapid nucleic acid hybridization techniques, in combination with improved cultural methods is the most promising of these approaches. Such methodological improvements should facilitate gathering data on important questions concerning the epidemiology of Listeria, and the natural history of listeriosis.

In vitro activities of aztreonam, piperacillin, and ticarcillin combined with amikacin against amikacin-resistant Pseudomonas aeruginosa and P. cepacia isolates from children with cystic fibrosis.

Amikacin, combined with aztreonam, piperacillin, or ticarcillin, synergistically inhibited amikacin-resistant sputum isolates of Pseudomonas aeruginosa and P. cepacia from children with cystic fibrosis. Ticarcillin-amikacin was the least active combination. Aminoglycoside resistance should not preclude the use of beta-lactam-aminoglycoside combinations in the treatment of pulmonary infections in cystic fibrosis.

In-vitro activity of ciprofloxacin and other antibacterial agents against Pseudomonas aeruginosa and Pseudomonas cepacia from cystic fibrosis patients.

The in-vitro activities of ciprofloxacin, a new oxyquinoline derivative, norfloxacin and six anti-pseudomal beta-lactam antibiotics were tested against pulmonary isolates of smooth and mucoid colony forms of Pseudomonas aeruginosa, and Ps. cepacia from children with cystic fibrosis. Ciprofloxacin was the most effective of the agents tested against either species. Minimal inhibitory concentrations of ciprofloxacin were 0.5, and 16 mg/l, for 90% of the Ps. aeruginosa and Ps. cepacia strains tested, respectively. No effect of inoculum size or discordance between inhibitory or bactericidal concentrations was observed. Ciprofloxacin is a potentially useful agent for the treatment of acute pseudomonal pulmonary exacerbations in children with cystic fibrosis.

Comparison of cefpiramide (HR-810) and four anti-pseudomonal beta-lactam agents against pseudomonas isolates from children with cystic fibrosis.

Cefpiramide (HR-810), ceftazidime, piperacillin, ticarcillin, and aztreonam were tested against tobramycin-sensitive and -resistant strains of Pseudomonas aeruginosa and tobramycin/amikacin-resistant isolates of Ps. cepacia recovered from the sputum of patients with cystic fibrosis. Against Ps. aeruginosa, none of the drugs inhibited 90% of the test strains at levels of less than 128 mg/l. Median minimal, inhibitory concentrations (MIC50) for all of the beta-lactam agents were lower for tobramycin-sensitive versus tobramycin-resistant isolates of Ps. aeruginosa. Ceftazidime was the most effective agent against Ps. cepacia. Aminoglycoside-resistance appears to be associated with significant beta-lactam resistance in Ps. aeruginosa isolated from the sputum of patients with cystic fibrosis.

Pseudomonas cepacia: biology, mechanisms of virulence, epidemiology.

Pseudomonas cepacia, originally described as a plant pathogen, has emerged as an important cause of infection in altered hosts, particularly in the hospital setting. This organism's ability to survive and proliferate in a variety of solutions, medications, and even disinfectants and antiseptics has resulted in numerous clusters of common-source nosocomial infections. Many patients exposed to P. cepacia are merely colonized, but serious infections, including surgical and burn wound infections, bacteremia, meningitis, pneumonia, peritonitis, and urinary tract infections, are not rare. The virulence properties of this pathogen remain poorly characterized. Recently, P. cepacia has been reported in some cystic fibrosis centers as an increasingly frequent pulmonary pathogen. This trend has caused considerable concern because of reports of occasional cases of fulminant necrotizing pneumonia and bacteremia. Conversely, many patients with CF who become colonized with this organism have no ill effects. The epidemiology of P. cepacia in the CF population is unclear, but some patients probably acquire the organism from colonized siblings with CF. Circumstantial evidence suggests that the organism may also be acquired in the hospital. Treatment of infections is exceedingly difficult, particularly in patients with CF, because P. cepacia is resistant to a broad range of antibiotics.

Cystic fibrosis. Infection and immunity to Pseudomonas.

Chronic pulmonary infection with P. aeruginosa in CF may result from: 1. An initial failure of clearance mechanisms (increased adherence) leading to the development of a highly compartmentalized inflammatory reaction; 2. Inhibition of clearing mechanisms for bacteria present in the bronchial lumen; and 3. A largely ineffective, and possibly damaging, hyperactivity of inflammatory cells in the lumen and bronchial wall. The special relationship between the CF host and P. aeruginos, always long-term, and frequently subtle in its complexity, needs further understanding in order to develop new strategies for the treatment of chronic lung infections with this organism.

Isolation and characterization of mutants of Escherichia coli K12 resistant to the new aminoglycoside antibiotic, amikacin.

Spontaneous mutants of Escherichia coli K12 that are resistant to the new aminoglycoside antibiotic, amikacin, were isolated. These mutants have simultaneously acquired cross-resistance to kanamycin, gentamicin and neomycin but not to streptomycin or spectinomycin. Sensitivity of the mutant strains to the non-aminoglycoside antibiotics, ampicillin, tetracycline and polymyxin, was unaffected. The mutation responsible for amikacin resistance was mapped by PI transduction and found to be tightly linked to strA, distal with respect to spcA and aroE.

Antibodies to proteases and exotoxin A of Pseudomonas aeruginosa in patients with cystic fibrosis: Demonstration by radioimmunoassay.

Sera from 33 patients with cystic fibrosis and two pediatric patients being treated for chronic pulmonary infections not related to cystic fibrosis and six sera or serum pools from uninfected individuals were tested with a microtiter radioimmunoassay for reactivity against exotoxin A and two proteases from Pseudomonas aeruginosa. Exotoxin A was purified from a low-protease strain of P. aeruginosa and shown to have adenosine diphosphate-ribose transferase activity and mouse lethality. Proteases were purified from an isolate of P. aeruginosa from a patient with cystic fibrosis and had proteolytic activity against elastin and collagen in an assay employing dimethylated protein substrates. The antibody responses of the patients detected using 125I-labeled antibody to human immunoglobulin were correlated with clinical evaluations expressed as a composite score based on pulmonary findings, case histories, growth and nutrition, and chest X rays. Values in the radioimmunoassay for patients' sera were compared with those of a control serum pool and expressed as the ratio of counts per minute (cpm) in patient serum to the cpm in the control pool. Inverse correlations were found between these ratios for each of the pseudomonas exoproducts and clinical scores; highest ratios occurred in patients showing the lowest clinical scores. These results confirm that proteases and exotoxin A of P. aeruginosa are produced in cystic fibrosis pulmonary infections due to P. aeruginosa and suggest that they may serve as significant virulence factors in these chronic infectious states.

Induction of phagocytic inhibitory activity in cats with chronic Pseudomonas aeruginosa pulmonary infection.

Chronic pulmonary infection has been established in cats by repeated intrapulmonary inoculation of viable Pseudomonas aeruginosa enmeshed in agarose beads. In the serum of all chronically infected animals, a substance(s) developed which inhibited phagocytosis of P. aeruginosa by normal cat alveolar macrophages. Phagocytosis was measured by incubating macrophage monolayers (5 X 10(5) alveolar macrophages) for 20 min in the presence of 3H-labeled bacteria and 5% serum from control or infected animals. Inhibitory activity developed 4 to 16 weeks after initial infection, and inhibition of phagocytosis of P. aeruginosa in the presence of infected cat serum ranged from 30 to 79%. After inhibitory activity developed, it persisted throughout the remainder of the experiment in each animal. The activity was specific for P. aeruginosa of the infecting serotype and did not affect phagocytosis of gram-positive organisms. Inhibitory activity was unchanged by heating serum at 56 degrees C for 30 min. We have previously described a P. aeruginosa-specific, heat-stable, phagocytosis-inhibitory activity in the serum of patients with cystic fibrosis. Since inhibitory activity also develops in cats with chronic P. aeruginosa pulmonary infection, such activity may not be a primary intrinsic abnormality in patients with cystic fibrosis. The animal model described here offers a system for following the development of and for characterization of the P. aeruginosa-specific phagocytosis-inhibitory activity.

Suppression of in vitro lymphocyte DNA synthesis by killed Pseudomonas aeruginosa.

Whole antibiotic-killed classic Pseudomonas aeruginosa organisms elicited human lymphocyte [3H]thymidine (TdR) uptake in vitro after 5 days in culture. However, high concentrations of the same preparation did not elicit [3H]TdR incorporation. The investigation of this lymphocyte unresponsiveness revealed that a high dose of P. aeruginosa, when added to lymphocyte cultures together with optimal concentrations of lymphocyte activators (e.g., plant lectins or whole killed Staphylococcus aureus Cowan 1), caused a potent, nonspecifically expressed inhibition of lymphocyte [3H]TdR uptake in response to these mitogens. High doses of P. aeruginosa were not cytotoxic to lymphocytes, and the inhibition caused was reversed when lymphocytes were washed free of bacteria. The inhibition of [3H]TdR uptake by high-dose P. aeruginosa did not require the generation of adherent suppressor cells or prostaglandin-mediated, steroid-sensitive or radiation-sensitive suppressor mechanisms. At optimal lymphocyte stimulatory concentrations of P. aeruginosa, the addition of indomethacin or the depletion of adherent cells caused an increase in lymphocyte [3H]TdR incorporation. This is consistent with an adherent-cell population regulating [3H]TdR uptake in response to P. aeruginosa via a prostaglandin-dependent pathway. This population was not involved in the inhibition of lymphocyte [3H]TdR uptake by high concentrations of P. aeruginosa.

Protective immunization against chronic Pseudomonas aeruginosa pulmonary infection in rats.

Rats were immunized systemically with various doses of the polyvalent Pseudomonas aeruginosa vaccine PEV-01. After a series of two or three doses (25 to 50 micrograms each) at 8- to 11-day intervals, animals were challenged intratracheally by the agarose bead technique with a serotype 5 P. aeruginosa strain at periods of 9 to 42 days. Immunized animals developed circulating antibodies (primarily immunoglobulin M) against vaccine components at levels significantly higher than challenged, nonimmunized controls (P less than 0.005). Eight to ten days postinfection, histological sections of lungs from immunized animals showed only minimal inflammation associated with infectious foci (agarose beads) as compared with the extensive pathological changes of airways and parenchyma seen in infected nonimmunized control animals. However, no significant reduction in bacterial numbers was observed. Such protection lasted at least 6 weeks after the final immunization. It is speculated that the vaccine may contain components of cell surface proteins and virulence exoproducts.