Pseudomonas aeruginosa

Clin Genet, 2002 Oct, 62(4), 325 - 7
Analysis of the two common alpha-1-antitrypsin deficiency alleles PiMS and PiMZ as modifiers of Pseudomonas aeruginosa susceptibility in cystic fibrosis; Meyer P et al.; Lung disease is the direct cause of death in more than 90% of cystic fibrosis (CF) patients . Proteinase-antiproteinase imbalances are common in CF and alpha-1-antitrypsin (AAT) deficiency . We investigated the hypothesis that the AAT deficiency alleles PiS and PiZ contribute to pulmonary prognosis in CF . Two hundred and sixty-nine CF patients from Southern Germany were included in this study . The serum concentrations of AAT and C-reactive protein (CRP) were determined by nephelometry, and patients were screened by polymerase chain reaction (PCR) and restriction enzyme digest for the common AAT deficiency alleles PiS and PiZ . The onset of chronic bacterial colonization by Pseudomonas aeruginosa (Pae) was correlated with the AAT phenotypes PiMM, PiMS and PiMZ . Only three out of nine CF patients (33%) diagnosed with either PiMS or PiMZ had developed chronic Pae lung infection earlier in their lives . The remaining six patients showing a PiMS or PiMZ phenotype showed a later onset of chronic Pae lung infection . Our results indicate that PiMS and PiMZ are not associated with worse pulmonary prognosis in CF . These data need to be confirmed in studies with a much larger number of cases.

J Immunol, 2002 Oct 15, 169(8), 4522 - 30
Pseudomonas aeruginosa activates human mast cells to induce neutrophil transendothelial migration via mast cell-derived IL-1 alpha and beta; Lin TJ et al.; The mechanisms of neutrophil (PMN) recruitment to Pseudomonas aeruginosa infection remain incompletely defined . Mast cells (MC) involvement in this process has not been studied previously . In this study, we demonstrate that human cord blood-derived MC phagocytose P . aeruginosa and release mediators that activate HUVEC monolayers for supporting PMN transmigration . Pretreatment of supernatants from P . aeruginosa-MC cocultures with neutralizing anti-IL-1alpha plus anti-IL-1beta Abs, or IL-1R antagonist before addition to HUVEC for stimulation completely abrogated MC-induced PMN transmigration, while anti-TNF-alpha treatment had no effect . The expression of E-selectin and ICAM-1 on HUVEC, the latter a ligand for PMN CD11/CD18, was significantly up-regulated by P . aeruginosa-induced MC mediators . Pretreatment of human PMN with anti-CD18 mAb or pretreatment of HUVEC with a combination of three mAbs (against ICAM-1, ICAM-2, and E-selectin) inhibited by 85% the MC-dependent PMN transmigration . Moreover, P . aeruginosa-induced production of IL-1alpha and IL-1beta was down-regulated by IL-10 and dexamethasone . This study demonstrates for the first time that MC may mediate P . aeruginosa-induced PMN recruitment via production of IL-1alpha and beta . These findings have important implications for diseases involving P . aeruginosa infection and suggest novel targets for modulating P . aeruginosa-induced inflammation.

Microbiology, 2002 Oct, 148(Pt 10), 3195 - 202
Physiological responses of Pseudomonas aeruginosa PAO1 to oxidative stress in controlled microaerobic and aerobic cultures; Sabra W et al.; Pseudomonas aeruginosa PAO1 was found to exhibit several remarkable physiological responses to oxidative stress upon its growth in a computer-controlled suspension culture . First, it strongly reduced the transfer rate of oxygen from the gas into the liquid phase, causing oxygen-limited or microaerophilic conditions in the culture after a short period of cultivation, even at high aeration rates with pure oxygen . Second, PAO1 that was previously classified as 'non-mucoid' formed a clear polysaccharide capsule on the cell surface (mucoid phenotype) under oxidative-stress conditions . Third, the strain showed a reduced growth rate and a longer lag phase under high oxygen tension . Finally, P . aeruginosa PAO1 released a high amount of proteins into the culture broth . The release of some virulence factors by PAO1, such as elastase, was significantly enhanced or only occurred under microaerobic conditions (i.e . dissolved oxygen tension value around 1% of air saturation) . Hence, it is concluded that P . aeruginosa PAO1 prefers microaerobic conditions for growth and for the formation of some of its virulence factors . PAO1 can create such growth conditions by at least two mechanisms: (i) blockage of the transfer of oxygen and (ii) formation of a polysaccharide capsule on the cell surface . It is postulated that the blockage of oxygen transfer may play an important role in the defence of this pathogen against reactive oxygen intermediates.

Microbiology, 2002 Oct, 148(Pt 10), 3183 - 93
The Pseudomonas aeruginosa alternative sigma factor PvdS controls exotoxin A expression and is expressed in lung infections associated with cystic fibrosis; Hunt TA et al.; PvdS is an alternative sigma factor regulated by the global iron regulator Fur . It has been demonstrated that PvdS plays a role in the iron-dependent regulation of exotoxin A (ETA) in Pseudomonas aeruginosa strain PAO1 . The goals of this research were to determine if pvdS was transcribed by the bacteria in the chronic lung infections associated with cystic fibrosis (CF) and to determine how PvdS interacts with the regAB promoters of the hyper-toxigenic strain PA103 . It was found that pvdS is transcribed in the lungs of patients with CF and that it appears to be involved with the regulation of toxA in this environment . This correlated with the finding that in strain PA103, a mutation in pvdS reduced ETA activity while the same mutation in strain PAO1 abrogated ETA production . It was also shown that in strain PA103, pvdS was absolutely required for activation of the regAB P2 promoter . The effect of PvdS on the P2 promoter may be direct or indirect; however, in support of a direct role, an eight-out-of-nine base-pair match to the consensus sequence for PvdS binding was identified at the transcriptional start site for the P2 promoter . The effect of PvdS on the PA103 regAB P1 promoter under aerobic growth conditions was also examined . The results show that PvdS does modulate the expression from this promoter but that both the regAB operon and PvdS are required for optimal P1 promoter activity . These studies demonstrate that the alternative sigma factor PvdS acts as a regulator of ETA expression in P . aeruginosa strain PA103 through the regAB operon and that PvdS is expressed in lung infections associated with CF.

Microbiology, 2002 Oct, 148(Pt 10), 2987 - 96
A polymorphic region in Mycobacterium abscessus contains a novel insertion sequence element; Howard ST et al.; A polymorphic region was discovered in the genetically uncharacterized opportunistic pathogen Mycobacterium abscessus . The region contains a novel 1.7 kb insertion sequence (IS) named ISMab1 . ISMab1 contains two complete ORFs and one partial ORF located in segments with over 80% nucleotide identity to Mycobacterium avium IS1601 and IS999 and to previously unreported IS-like elements from Mycobacterium smegmatis . The marked similarity within this family of elements is supportive of horizontal transfer between environmental mycobacterial species . In clinical isolates, ISMab1 was either present as a single copy or absent . The polymorphic region containing ISMab1 was identified by genomic subtraction between a parental strain and phenotypic variant . The variant has a 14.2 kb genomic deletion and this is flanked in the parental strain by complex arrays of inverted and direct repeats . Clinical isolates of M . abscessus were probed for the deletion and flanking sequences and two were found to be missing more than 20 kb . No regional deletions were found in the type strain, ATCC 19977 . Although M . abscessus is a rapidly growing species, comparative sequence analysis of 23 kb from the polymorphic region showed that most local ORFs have greater amino acid identity to proteins encoded by genes from the slowly growing mycobacteria, M . avium and Mycobacterium tuberculosis, than to the rapid-grower M . smegmatis . Several ORFs also have strong similarity to Pseudomonas aeruginosa genes with a potential role in beta-oxidation.

Clin Exp Optom, 2002 Sep, 85(5), 271 - 8
The pathogenesis of bacterial keratitis: studies with Pseudomonas aeruginosa; Fleiszig SM et al.; Bacterial keratitis is a sight-threatening corneal disease that is most commonly associated with the extended wear of soft contact lenses . Over the past decade, we have investigated the pathogenesis of infectious keratitis involving the opportunistic pathogen Pseudomonas aeruginosa . Our research has focused on understanding the respective roles of bacteria and host in the establishment of this infection . Here, we provide a current perspective on P . aeruginosa keratitis, reviewing some of the research developments that have helped shape our views on the mechanisms by which pathogen and host response cause corneal disease . P . aeruginosa may provide a model for the pathogenesis of bacterial keratitis and help further elucidate the complex array of host factors that normally protect the cornea from infectious agents.

J Enzyme Inhib Med Chem, 2002 Feb, 17(1), 1 - 7
Antibacterial Schiff bases of oxalyl-hydrazine/diamide incorporating pyrrolyl and salicylyl moieties and of their zinc(II) complexes; Chohan ZH et al.; Schiff bases derived from oxaldiamide/oxalylhydrazine and pyrrol-2-carbaldehyde, or salicylaldehyde respectively, as well as their Zn(II) complexes have been prepared and tested as antibacterial agents . These Schiff bases function as tetradentate ligands, forming octahedral Zn(II) complexes . The ketonic form for the diamide derived Schiff base and the enolic form of the hydrazide derived Schiff base were the preferred tautomers for coordination of the metal ions . The title compounds and their Zn(II) derivatives were evaluated for antibacterial activity against several bacterial strains which easily develop resistance to classical antibiotics, such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa . Some of them showed promising biological activity in inhibiting the growth of such organisms.

Proteomics, 2002 Sep, 2(9), 1325 - 46
Proteomic comparison of membrane and extracellular proteins from invasive (PAO1) and cytotoxic (6206) strains of Pseudomonas aeruginosa; Nouwens AS et al.; Strains of Pseudomonas aeruginosa can be phenotypically classified by their mode of pathogenicity as either invasive, where the bacterium is internalised by host cells, or cytotoxic, where the host cell is killed without internalisation through the expression of cytotoxicity factors . These phenotypes are thought to depend primarily on the interactions of pseudomonal membrane and secreted proteins with host cells . We report here comparisons of outer membrane and extracellular protein-enriched fractions from invasive (PAO1) and cytotoxic (6206) strains of P . aeruginosa separated by two-dimensional (2-D) gel electrophoresis . Gel image comparisons revealed the two strains express essentially identical membrane protein profiles under the conditions investigated . Membrane protein strain differences were typically the result of minor amino acid sequence variations resulting in small mass and isoelectric point shifts visible on 2-D gels . Analysis of extracellular proteins from stationary phase growth, however, revealed significantly different protein profiles . Extracellular fractions from the invasive PAO1 strain were dominated by extracellular proteases including elastase (LasB), LasA protease and chitin-binding protein, as well as several previously designated 'hypothetical' proteins . LasB appeared to be highly processed with 28 discrete mass and isoelectric point forms detected in this study . The significant number of active extracellular proteases (including LasB itself) may account for this processing . Conversely, extracellular fractions from strain 6206 consisted mainly of cellular and membrane exposed proteins including GroEL, DnaK and flagellar subunits . These are thought to result from cellular turnover during growth and the reliance on the secretory mechanisms of this strain to produce high levels of cytotoxicity factors, such as ExoU, which may be produced only upon specific interactions with host cells . These studies will aid in elucidating the differences between invasive and cytotoxic P . aeruginosa at the proteome level.

J Cutan Med Surg, 2003 Jan-Feb, 7(1), 1 - 6 Epub 2002 Oct 09.
Barrier and antibacterial properties of 2-octyl cyanoacrylate-derived wound treatment films; Mertz PM et al.; BACKGROUND: Besides enhancing healing, an ideal dressing should prevent invasion of pathogens and control the number of bacteria already present in the wounds . OBJECTIVE: To evaluate the barrier and antimicrobial properties of a cyanoacrylate-based bandage (LAB) against Staphylococcus aureus or Pseudomonas aeruginosa on partial thickness wounds in swine . METHODS: Barrier study: Bacteria were inoculated over test materials (LAB, standard bandage, air-exposed) that were placed over wounds . The bacteria from wounds were quantitated at 24, 48, and 72 hours postinoculation . Antimicrobial study: Wounds inoculated with bacteria were covered with LAB, standard bandage, or hydrocolloid bandage or left air-exposed . The bacteria recovered from wounds were quantitated at 24 and 72 hours after treatment . RESULTS: Barrier study: No bacteria were recovered from LAB-treated wounds . Antimicrobial study: LAB reduced the number of inoculated bacteria in comparison to all other groups . CONCLUSION: LAB is effective in protecting wounds from external bacterial invasion and reducing bacterial contamination.

Am J Respir Crit Care Med, 2002 Oct 1, 166(7), 988 - 93
Epidemiology of Pseudomonas aeruginosa in cystic fibrosis in British Columbia, Canada; Speert DP et al.; Pseudomonas aeruginosa is the most common respiratory pathogen in patients with cystic fibrosis (CF), but the predominant mechanism by which it is acquired is controversial . To determine the frequency of patient-to-patient spread, we evaluated P . aeruginosa isolates from 174 patients treated at the CF clinics in Vancouver, BC, Canada, since 1981 . Multiple isolates were obtained from each patient and genetically typed by random amplified polymorphic DNA and pulsed field gel electrophoresis analyses . A total of 157 genetic types of P . aeruginosa was identified, 123 of which were unique to individual patients . A total of 34 types was shared by more than one patient; epidemiologic evidence linked these individuals only in the cases of 10 sibships and 1 pair of unrelated patients . We conclude that there is an extremely low risk in Vancouver for patients with CF to acquire P . aeruginosa from other patients . It appears that prolonged close contact, such as occurs between siblings, is necessary for patient-to-patient spread . The major source of acquisition of P . aeruginosa in CF appears to be from the environment . Considering these observations, we do not recommend segregation of patients with CF on the basis of their colonization status with P . aeruginosa.

Am J Respir Crit Care Med, 2002 Oct 1, 166(7), 983 - 7
Detection of a widespread clone of Pseudomonas aeruginosa in a pediatric cystic fibrosis clinic; Armstrong DS et al.; Cross-infection by Pseudomonas aeruginosa between unrelated patients with cystic fibrosis (CF) is believed to be uncommon . After detecting a genotypically identical strain of P . aeruginosa in five unrelated children with CF dying from severe lung disease, we determined its prevalence within a large CF clinic using pulsed-field gel electrophoresis and random amplified polymorphic DNA assays . The clinical status of P . aeruginosa-infected patients was also determined . Between September and December 1999, 152 patients, aged 3.9-20.7 years, provided sputum for culture . P . aeruginosa was detected in 118 children of mean (SD) age 13.5 (3.8) years . The genotyping techniques were concordant, showing that 65 (55%) infected patients carried an indistinguishable or closely related strain . No distinctive antibiogram or environmental reservoir was found . Patients with the clonal strain were more likely than those with unrelated isolates to have been hospitalized in the preceding 12 months for respiratory exacerbations . This study demonstrates extensive spread of a single, clonal strain of P . aeruginosa in a large pediatric CF clinic . Whether this strain is also more virulent than sporadic isolates remains to be determined . As transmissible strains could emerge elsewhere, other CF clinics may also need to consider molecular methods of surveillance for cross-infection.

Vet Dermatol, 2002 Oct, 13(5), 247 - 51
Isolation and characterization of Pseudomonas aeruginosa from sheep with fleece rot in northern and middle Jordan; El-Sukhon SN; A total of 162 sheep fleece samples were collected from 2315 sheep clinically examined for evidence of dermatitis . The sheep belonged to 32 flocks raised in northern and middle Jordan . Eighty-three samples showed no obvious abnormalities, whereas the remainder showed exudation (79 samples), fleece discoloration (18) and fleece roughness (40) and abscesses (7) . Seventeen Pseudomonas aeruginosa isolates were obtained from these samples . Antibacterial resistance in vitro was common; resistance to tetracycline, amoxycillin, erythromycin and cotrimoxazole was shown by > 90% of the isolates . Resistance to norfloxacin (29.4% of isolates), ciprofloxacin (17.6%) and amikacin (17.6%) was also demonstrated . Fourteen isolates were serum resistant when assessed after 1-3 h incubation in sheep and calf sera, and their count increased by 2-2.9 and 2.5-3.5 respectively.

Eur Respir J, 2002 Sep, 20(3), 658 - 64
A randomised clinical trial of nebulised tobramycin or colistin in cystic fibrosis; Hodson ME et al.; Chronic infection with Pseudomonas aeruginosa is associated with progressive deterioration in lung function in cystic fibrosis (CF) patients . The purpose of this trial was to assess the efficacy and safety of tobramycin nebuliser solution (TNS) and nebulised colistin in CF patients chronically infected with P . aeruginosa . One-hundred and fifteen patients, aged > or = 6 yrs, were randomised to receive either TNS or colistin, twice daily for 4 weeks . The primary end point was an evaluation of the relative change in lung function from baseline, as measured by forced expiratory volume in one second % predicted . Secondary end points included changes in sputum P . aeruginosa density, tobramycin/colistin minimum inhibitory concentrations and safety assessments . TNS produced a mean 6.7% improvement in lung function (p=0.006), whilst there was no significant improvement in the colistin-treated patients (mean change 0.37%) . Both nebulised antibiotic regimens produced a significant decrease in the sputum P . aeruginosa density, and there was no development of highly resistant strains over the course of the study . The safety profile for both nebulised antibiotics was good . Tobramycin nebuliser solution significantly improved lung function of patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa, but colistin did not, in this study of 1-month's duration . Both treatments reduced the bacterial load.

Pediatr Pulmonol, 2002 Nov, 34(5), 336 - 41
Do in-line respiratory filters protect patients? Comparing bacterial removal efficiency of six filters; Canakis AM et al.; With all pulmonary function diagnostic and respiratory therapy equipment, cross-infection has always been a concern, especially in the cystic fibrosis population, in whom pulmonary function tests are done routinely . The aim of this study was to identify and compare the bacterial removal efficiency (BRE, ability of a filter to remove microorganisms) of six different filters used in hospital settings: Microgard (MG), Spirobac (SB), PALL (PL), and KOKO (KK), used in the pulmonary function laboratory; and Clear-Guard (CG) and Respigard (RG), used in ventilator circuits . Filters were tested in both saturated and nonsaturated conditions . A Pseudomonas aeruginosa suspension of 1 x 10(4) to 1 x 10(8) CFU/mL was nebulized onto each filter . A blood agar plate was held immediately downstream from the filter . Colony-forming units (CFU) were then counted after 24 hr of incubation . A peak flow was applied across the spirometry filters . Bacterial thresholds of the filters were also identified (concentration of bacteria at which a filter no longer has 100% BRE) . There was a significant difference in BRE among the six filters in saturated states when challenged with 1 x 10(4) CFU/mL (MG, KK, CG, and RG, 100%; SB, 98.8%; PL, 42.7%; P = 0.003) . There was no significant difference between saturated and nonsaturated states, or after application of a peak flow . Filter thresholds were significantly different (KK 1 x 10(8), MG 1 x 10(7), CG 1 x 10(6), RG 1 x 10(5), and SB and PL <1 x 10(4) CFU/mL) . In conclusion, when all filters are exposed to the same extreme challenges, significant differences exist in their ability to remove bacteria .

J Antimicrob Chemother, 2002 Oct, 50(4), 479 - 86
Evaluation of media available for testing the susceptibility of Pseudomonas aeruginosa by BSAC methodology; Andrews J et al.; BSAC methodology was used to study media available from four manufacturers for susceptibility testing of Pseudomonas aeruginosa in the UK and Ireland . Fifty isolates of P . aeruginosa, including ATCC and NCTC control strains, were studied by disc testing methods by 20 centres around the UK and Ireland and by MIC determination at the BSAC Standardized Method Development Centre (SMDC) in Birmingham, UK . Although many of the antibiotics tested gave similar zone diameters for a particular strain-antibiotic combination irrespective of the medium used for testing, significant differences in media performance were noted for the aminoglycosides, imipenem and colistin . The data generated showed that for testing aminoglycosides, zone diameters on two of the agars were significantly larger than those on Oxoid IsoSensitest agar-the recommended medium for the BSAC method . For imipenem, zones were smaller on one of the agars and for colistin, zones were larger on one of the agars in comparison with those on Oxoid agar . Scattergram analysis of zone diameters and MICs of colistin indicated that the zone diameter breakpoints should be adjusted . Media purported to be equivalent to Oxoid IsoSensitest agar do not give the same zone sizes with some agents and should not be used until standards for media performance have been established and the media have been shown to perform adequately.

Tuberculosis (Edinb), 2002, 82(2-3), 85 - 90
Is Mycobacterium tuberculosis a closer relative to Gram-positive or Gram-negative bacterial pathogens?
Fu LM, Fu-Liu CS.
The phylogenetic position of Mycobacterium tuberculosis relative to other bacteria is controversial . Its cell wall has characteristics of both Gram-positive and Gram-negative bacteria . In the standard reference of bacterial phylogeny based on 16S ribosomal RNA sequence comparison, M . tuberculosis belongs to the high G+C Gram-positive bacteria that form a monophyletic group with the low G+C Gram-positive bacteria such as Bacillus subtilis . Some analyses indicate no particular relationship between these two groups . The availability of the complete genome sequence of M . tuberculosis allows us to reexamine this issue from genomic perspectives, as genome-based phylogenies may be more representative of the evolutionary history of whole organisms than molecular trees . In the genome tree constructed based on conserved gene content, M . tuberculosis is more related to Gram-negative than to Gram-positive bacteria as reflected by the evolutionary distance between nearest ancestral units . This conclusion may be supported by another analysis showing that M . tuberculosis shares relatively more orthologous genes for energy production and conversion with Gram-negative bacteria, in particular, Escherichia coli and Pseudomonas aeruginosa, than with Gram-positive bacteria.

Eur J Biochem, 2002 Oct, 269(19), 4738 - 45
The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel; Yoshihara E et al.; OprM, the outer membrane component of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa, has been assumed to facilitate the export of antibiotics across the outer membrane of this organism . Here we purified to homogeneity the OprM protein, reconstituted it into liposome membranes, and tested its channel activity by using the liposome swelling assay . It was demonstrated that OprM is a channel-forming protein and exhibits the channel property that amino acids diffuse more efficiently than saccharides . However, antibiotics showed no significant diffusion through the OprM channel in the liposome membrane, suggesting that OprM functions as a gated channel . We reasoned that the protease treatment may cause the disturbance of the gate structure of OprM . Hence, we treated OprM reconstituted in the membranes with alpha-chymotrypsin and examined its solute permeability . The results demonstrated that the protease treatment caused the opening of an OprM channel through which antibiotics were able to diffuse . To elucidate which cleavage is intimately related to the opening, we constructed mutant OprM proteins where the amino acid at the cleavage site was replaced with another amino acid . By examining the channel activity of these mutant proteins, it was shown that the proteolysis at tyrosine 185 and tyrosine 196 of OprM caused the channel opening . Furthermore, these residues were shown to face into the periplasmic space and interact with other component(s) . We considered the possible opening mechanism of the OprM channel based on the structure of TolC, a homologue of OprM.

Shock, 2002 Sep, 18(3), 236 - 41
Heparin nebulization attenuates acute lung injury in sepsis following smoke inhalation in sheep; Murakami K et al.; Pseudomonas pneumonia is a common complication of smoke inhalation injury . Airway casts formed from clotted mucous occur frequently in this condition . A recent report shows that intravenous heparin improves oxygenation and reduces lung damage in a sheep model of smoke inhalation . We hypothesized that nebulized heparin could be an effective means of reducing cast formation . Female sheep (n = 19) were surgically prepared for a study of acute lung injury (ALI) . After a tracheotomy, 48 breaths of cotton smoke (<40 degrees C) were inflated into the airway . Afterwards, live Pseudomonas aeruginosa (5 x 10(11) CFU) was instilled into the lung . All sheep were mechanically ventilated with 100% O2 and were divided into four groups: a heparin-nebulized group (n = 5; animals received aerosolized heparin {10,000 I.U.} 1 h after the bacterial instillation and subsequently every 4 h thereafter), an intravenous heparin group (n = 5,300 U/kg/23 h, infusion was started 1 h after the injury), a saline-nebulization group (n = 5; animals received inhaled nebulized saline), and a sham injury group (n = 4, treated in the same fashion, but no injury) . The animals were sacrificed after 24 h of mechanical ventilation, and lung samples were harvested . Sheep exposed to lung injury presented with typical hyperdynamic cardiovascular changes and a corresponding drop in PaO2 . These changes were significantly attenuated in the heparin groups . Histological changes consisting of cellular infiltrates, lung edema, congestion, and cast formation were reduced by heparin . These data suggest that nebulized inhaled heparin is a beneficial therapy for sepsis-induced ALI.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 2), 1874 - 5 Epub 2002 Sep 28.
Crystallization and preliminary X-ray crystallographic analysis of peptide deformylase from Pseudomonas aeruginosa; Kim HW et al.; Peptide deformylase (PDF) from the pathogenic bacterium Pseudomonas aeruginosa has been overexpressed in Escherichia coli and crystallized in the presence of its inhibitor actinonin at 297 K using polyethylene glycol (PEG) 4000 as a precipitant . The diffraction limit and the spot shape of the crystals could be slightly improved by the crystal annealing/dehydration procedure . X-ray diffraction data to 1.85 A have been collected using synchrotron radiation . The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 68.75, b = 74.46, c = 77.18 A . The asymmetric unit contains two subunits of peptide deformylase, with a corresponding crystal volume per protein mass (V(M)) of 2.45 A(3) Da(-1) and a solvent content of 49.8%.

FEMS Microbiol Lett, 2002 Sep 10, 214(2), 217 - 22
Metabolic engineering of Escherichia coli for the production of medium-chain-length polyhydroxyalkanoates rich in specific monomers; Park SJ et al.; The Escherichia coli fabG(Ec) gene and the Pseudomonas aeruginosa rhlG(Pa) gene, which encode 3-ketoacyl-acyl carrier protein reductase, were expressed in E . coli W3110 and its fadA mutant strain WA101 to examine their roles in medium-chain-length (MCL) polyhydroxyalkanoate (PHA) biosynthesis from fatty acids . When one of these 3-ketoacyl-acyl carrier protein reductase genes was co-expressed with the Pseudomonas sp . 61-3 PHA synthase gene (phaC2(Ps)) in E . coli W3110, MCL-PHA composed mainly of 3-hydroxyoctanoate and 3-hydroxydecanoate was synthesized from sodium decanoate . When the fabG(Ec) gene and the phaC2(Ps) gene were co-expressed in the fadA mutant E . coli strain WA101, MCL-PHA rich in 3-hydroxydecanoate monomer up to 93 mol% was accumulated from sodium decanoate . This was possible by efficiently redirecting 3-ketoacyl-coenzymes A from the beta-oxidation pathway to the PHA biosynthesis pathway without losing two carbon units, the strategy of which can be extended for the production of MCL-PHAs rich in other specific monomers.

Appl Environ Microbiol, 2002 Oct, 68(10), 5096 - 103
Variability in Pseudomonas aeruginosa lipopolysaccharide expression during crude oil degradation; Norman RS et al.; Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil . To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared . Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells . Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation . U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset . Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells . The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation.

Biomaterials, 2002 Dec, 23(23), 4565 - 72
Polyclonal human antibodies reduce bacterial attachment to soft contact lens and corneal cell surfaces; Rediske AM et al.; Bacterial keratitis due to Pseudomonas aeruginosa is a potentially serious complication of extended-wear contact lens use . Adhesion of P . aeruginosa to soft contact lens materials or corneal endothelial cells in the presence of pooled human immunoglobulins and/or neutrophils in artificial tear fluid was studied in vitro as a potential method to treat contact lens-associated infection . Soft hydrophilic contact lens materials equilibrated in sterile saline were soaked in artificial tear fluid for 18 h prior to use . P . aeruginosa IFO 3455 was added to groups of lenses or confluent cultured bovine corneal endothelial cells with varying amounts of human polyclonal immunoglobulin (IgG) and human blood neutrophils or serum albumin as a control . After 2 or 4 h incubation, adherent viable bacteria on lenses were quantified . Fluorescence microscopy was used to assess bacterial adherence to bovine corneal endothelial cells in the presence and absence of IgG and neutrophils . Various concentrations of albumin had no effect on adhesion . Human immunoglobulin solutions (25 mg/ml) reduced P . aeruginosa adhesion by nearly 1 log and 2 logs after 2 and 4 h incubations, respectively . Neutrophils in combination with 25 mg/ml IgG reduced bacterial adhesion approximately 1 log over reduction in adhesion by neutrophils alone . Diluted human IgG (10 mg/ml) did not significantly decrease bacterial adhesion after 2 or 4 h, but did reduce adhesion in combination with human neutrophils at both time points . Similar reductions in amounts of fluorescently labeled bacteria adhered to cultured monolayers of corneal endothelial cells under these conditions were qualitatively observed.

Hepatology, 2002 Oct, 36(4 Pt 1), 918 - 26
Analysis of TCR antagonism and molecular mimicry of an HLA-A0201-restricted CTL epitope in primary biliary cirrhosis; Kita H et al.; Although the etiology and mechanism of primary biliary cirrhosis (PBC) is unknown, growing evidence suggests a major role for T cells . We have recently identified the first CD8 T-cell epitope, amino acid 159-167 of the E2 component of pyruvate dehydrogenase complexes (PDC-E2) . To seek for analogue peptide-antagonizing effector function of CTLs specific for this autoantigen, we examined the effector functions of the PDC-E2-specific CTLs against alanine substituted peptides . Furthermore, because molecular mimicry has been postulated as a possible cause of initiating PBC, we carried out studies aimed at identifying naturally occurring peptides for the 159-167 peptide of PDC-E2 that may serve as agonists . An alanine substitution at position 5 of this epitope significantly reduced peptide-specific effector functions of CTLs . Moreover, this analogue peptide inhibited effector functions of the CTLs to the prototype peptide, including cytotoxicity and IFN-gamma production . We also identified a peptide derived from Pseudomonas aeruginosa, which showed a higher binding affinity to the HLA-A*0201 than the prototype peptide . This homologous peptide was recognized by CTLs specific for the prototype epitope on PDC-E2 . In conclusion, a modification of the immunodominant autoepitope can be utilized to manipulate the CD8 T-cell responses against the autoantigen PDC-E2 . Our finding also supports the thesis that molecular mimicry may be implicated in the initiation of the autoreactive CD8 T-cell responses and has implications for the use of such peptides for immunotherapy.

Cell Physiol Biochem, 2002, 12(4), 207 - 14
Pseudomonas aeruginosa triggered apoptosis of human epithelial cells depends on the temperature during infection; Fillon S et al.; The induction of apoptosis is a hallmark in many bacterial infections . Here, we show that the environmental temperature during infection influences P . aeruginosainduced apoptosis in human conjunctiva epithelial Chang cells . Infection with the clinical isolate P . aeruginosa762 at 37 degrees C resulted in a rapid induction of apoptosis within 30 min . Apoptosis was mediated by upregulation of CD95-receptor expression on the surface of the infected cells and depolarisation of the mitochondrial membrane potential (DeltaPhim) as determined by FACS-analysis of immunostained cells or of cells loaded with the potential sensitive dye JC1 respectively . In contrast, P . aeruginosafailed to induce significant apoptosis and mitochondrial alterations at 22 degrees C . The loss of the apoptotic response at 22 degrees C correlated with the failure of the epithelial cells to upregulate CD95, while bacterial growth or secretion of bacterial exotoxins into the culture supernatant appeared to be not affected . From our data we conclude that P . aeruginosa762 induced apoptosis is dependent on the temperature during infection and that cellular pathways leading to CD95-upregulation are inhibited at low temperature .

J Biol Chem, 2002 Nov 29, 277(48), 46669 - 75 Epub 2002 Sep 20.
Insight into the catalytic mechanism of Pseudomonas aeruginosa exotoxin A . Studies of toxin interaction with eukaryotic elongation factor-2; Armstrong S et al.; The molecular nature of the protein-protein interactions between the catalytic domain from Pseudomonas aeruginosa exotoxin A (PE24H) and its protein substrate, eukaryotic elongation factor-2 (eEF-2) were probed using a fluorescence resonance energy transfer method . Single cysteine mutant proteins of PE24H were prepared and site-specifically labeled with the donor fluorophore IAEDANS (5-(2-iodoacetylaminoethylamino)-1-napthalenesulfonic acid), whereas eEF-2 was labeled with the acceptor fluorophore fluorescein . The association was found to be independent of ionic strength and of the co-substrate, NAD(+) but dependent upon pH . The lack of requirement for NAD(+) to produce the toxin-eEF-2 complex demonstrates that the catalytic process is a random order mechanism, thereby disputing the current model . The previously observed pH dependence for catalytic function can be assigned to the toxin-eEF-2 binding event, as the pH dependence of binding observed in this study showed a strong correlation with enzymatic activity . The ability of the toxin to bind eEF-2 with bound GTP/GDP was assessed using nonhydrolyzable analogues . The results from the substrate binding and catalytic activity experiments indicate that PE24H is able to interact and bind with eEF-2 in all of its guanyl nucleotide-induced conformational states . Thus, the toxin ribosylates eEF-2 regardless of the nucleotide-charged state of eEF-2 . These results represent the first detailed characterization of the molecular details and physiological conditions governing this protein-protein interaction.

J Bacteriol, 2002 Oct, 184(20), 5633 - 40
Divergent structure and regulatory mechanism of proline catabolic systems: characterization of the putAP proline catabolic operon of Pseudomonas aeruginosa PAO1 and its regulation by PruR, an AraC/XylS family protein; Nakada Y et al.; Pseudomonas aeruginosa PAO1 utilizes proline as the sole source of carbon and nitrogen via a bifunctional enzyme (the putA gene product) that has both proline dehydrogenase (EC 1.5.99.8) and pyrroline 5-carboxylate dehydrogenase (EC 1.5.1.12) activities . We characterized the pruR-putAP loci encoding the proline catabolic system of this strain . In contrast to the putA and putP (encoding proline permease) genes of other gram- negative bacteria, which are located at divergent or separate loci, Northern blotting demonstrated that the two genes form an operon in strain PAO1 . While the phylogenetic lineage of the PutP protein of strain PAO1 was related to that of the origin (80% identity to the P . putida counterpart), PutA of PAO1 (PutA(PAO)) was rather distantly related (47% identity) to the P . putida counterpart . Moreover, unlike the PutA proteins of P . putida and enteric bacteria, PutA(PAO) appeared to lack a regulatory function . Upstream of the putAP operon, the divergent PA0781 gene specified a hypothetical outer membrane protein with a molecular weight of 74,202 . This gene appeared to be dispensable for proline utilization as indicated by the normal growth of a knockout mutant of PA0781 on medium containing proline . The pruR (proline utilization regulator) gene immediately upstream of PA0781 encoded a transcriptional activator of the AraC/XylS protein family and mediated the proline-responsive expression of putAP . Primer extension studies identified a PruR-dependent promoter responsive to proline in the 5'-flanking region of putA . Thus, the proline utilization system of P . aeruginosa differs from that of P . putida with respect to putA structure, the organization of the putAP genes, and the regulatory mechanism of putA expression.

Clin Sci (Lond), 2002 Oct, 103(4), 417 - 24
Cystic fibrosis transmembrane conductance regulator (CFTR) activity in nasal epithelial cells from cystic fibrosis patients with severe genotypes; Andersson C et al.; Cystic fibrosis is a heterogenic disease, in which the phenotype can also vary for patients with the same genotype . In the present study the function of the cystic fibrosis transmembrane conductance regulator (CFTR) in nasal epithelial cells from 19 adult patients with cystic fibrosis was investigated . All patients had severe mutations, whereby no or little functional CFTR is expected in the plasma membrane . Of the patients, 15 were homozygous for deltaF508-CFTR (i.e . CTFR lacking residue Phe-508) . The others were deltaF508-heterozygous with 3659delC, 394delTT or 2183AA-->G . Nasal epithelial cells, obtained by nasal brushings, were loaded with the fluorescent probe N -(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide to measure Cl(-) efflux . In most of the cystic fibrosis patients, forskolin plus isobutylmethylxanthine was unable to elicit any response . Unexpectedly, cells from three cystic fibrosis patients (two deltaF508/deltaF508 patients and one deltaF508/3659delC patient) responded to stimulation in a wild-type manner . It was investigated whether this residual chloride transport function was associated with a milder phenotype . Clinical parameters studied were lung function, number of antibiotic courses, Shwachman score, Bhalla score, age at chronic colonization with Pseudomonas aeruginosa and the pattern of essential fatty acids in serum phospholipids . Unknown factors may affect the presence of functional CFTR in patients with severe CFTR mutations . However, we could not find a correlation between the response to cAMP and any of the phenotype parameters . It appears that functional cAMP transport in the nasal epithelium is no guarantee of a mild phenotype and, conversely, that a patient lacking cAMP-dependent chloride transport can develop a mild phenotype.

Indian J Med Res, 2002 Apr, 115, 153 - 7
Prevalence of extended spectrum beta lactamase producing gram negative bacteria in a tertiary care hospital; Mathur P et al.; BACKGROUND & OBJECTIVES: Extended spectrum beta lactamase (ESBL) producing Gram negative bacteria are increasingly being associated with hospital infections thereby rendering all beta lactams, except carbapenems ineffective in the treatment of infections related to these organisms . A knowledge about their prevalence is essential to guide the appropriate antibiotic treatment of severe infections in hospitalized patients . The present work was carried out to study the prevalence of ESBL producing Gram negative bacteria in a tertiary care hospital . METHODS: A total of 678 Gram negative bacteria were included in the study . They were isolated from various clinical samples obtained from indoor patients admitted to the All India Institute of Medical Sciences, New Delhi during March to June 2001 . These isolates were screened for ESBL production by the inhibitor based test recommended by the National Committee for Clinical Laboratory Standards (NCCLS) using Klebsiella pneumoniae ATCC 700603 as positive control and Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 as negative controls . RESULTS: Out of the 678 strains tested, 458 (68%) were found to be ESBL producers . Among the bacterial species, ESBL production was most common in Klebsiella spp . (80%) . The proportion of ESBL positive isolates was highest from intensive care units (79%), followed by Medical Oncology (75%), Medical (54%) and Surgical wards (50%) . INTERPRETATION & CONCLUSION: A high prevalence of ESBL positive isolates was found in our hospital . This has important implications as carbapenems remain the only choice of treatment for infections caused by these organisms . The control measures include judicious use of antibiotics and implementation of appropriate infection control measures to control the spread of these strains in the hospital.

J Virol, 2002 Oct, 76(20), 10437 - 43
Bronchoalveolar fluid is not a major hindrance to virus-mediated gene therapy in cystic fibrosis; Rooney CP et al.; Successfully targeting the airway epithelium is essential for gene therapy of some pulmonary diseases . However, the airway epithelium is resistant to virus-mediated gene transfer with commonly used vectors . Vectors that interact with endogenously expressed receptors on the apical surface significantly increase gene transfer efficiency . However, other endogenous components involved in host immunity may hinder virus-mediated gene transfer . We tested the effect of bronchoalveolar lavage liquid (BAL) from patients with cystic fibrosis (CF), BAL from subjects without CF (non-CF BAL), Pseudomonas aeruginosa-derived proteins, and an array of inflammatory proteins on gene transfer mediated by adeno-associated virus type 5 (AAV5) and adenovirus targeted to an apically expressed glycosylphosphatidylinositol-modified coxsackie-adenovirus receptor . We found that neither CF BAL nor its components had a significant effect on gene transfer to human airway epithelium by these vectors . Non-CF BAL significantly impaired adenovirus-mediated gene transfer . Removal of immunoglobulins in non-CF BAL restored gene transfer efficiency . As virus vectors are improved and mechanisms of humoral immunity are elucidated, barriers to successful gene therapy found in the complex environment of the human lung can be circumvented.

Vet Ophthalmol, 2002 Sep, 5(3), 217 - 20
Squamous cell carcinoma associated with a periorbital mass in a veiled chameleon (Chamaeleo calyptratus); Abou-Madi N et al.; This report describes a squamous cell carcinoma in a 1-year-old female veiled chameleon (Chamaeleo calyptratus) . The lesion developed as a small (1 by 1 mm) left periocular discoloration of a scale never involving the eye . The mass was first diagnosed as an abscess, increased in size (4 by 8 by 3 mm), and recurred after two surgical resections combined with antibiotic therapy . Poor nutritional condition and egg production by the chameleon complicated management of this condition . The mass was removed surgically a third time at which point histopathologic evaluation revealed a locally invasive squamous cell carcinoma . Bacterial culture of the mass isolated a pure culture of Pseudomonas aeruginosa . Ceftazidime was administered at 20 mg/kg IM every 48 h for 20 days . The animal died 3 months later from complications during an ovariohysterectomy for pregnancy toxemia and oviduct inertia . Necropsy showed no local recurrence or metastasis of the tumor.

Antimicrob Agents Chemother, 2002 Oct, 46(10), 3286 - 7
Carbapenem-resistant Pseudomonas aeruginosa in malaysia producing IMP-7 beta-lactamase; Ho SE et al.; We have isolated and identified a carbapenem-resistant Pseudomonas aeruginosa strain from Malaysia that produces an IMP-7 metallo-beta-lactamase . This isolate showed high-level resistance to meropenem and imipenem, the MICs of which were 256 and 128 micro g/ml, respectively . Isoelectric focusing analyses revealed pI values of >9.0, 8.2, and 7.8, which indicated the possible presence of IMP and OXA . DNA sequencing confirmed the identity of the IMP-7 determinant.

Res Microbiol, 2002 Jul-Aug, 153(6), 339 - 44
Alkane biodegradation in Pseudomonas aeruginosa strains isolated from a polluted zone: identification of alkB and alkB-related genes; Belhaj A et al.; Pseudomonas aeruginosa strains that grow on crude oil as the sole source of carbon and energy were isolated from an environment in Morocco polluted by petroleum refinery effluents . The twenty isolates grew on saturated alkanes from C12 to C22 . Three of the isolates were also able to grow on low molecular weight C6 to C10 n-alkanes, but the other 17 strains were not . The strains were tested for alkB and a/kB-related genes encoding alkane-1-monooxygenase (alkane hydroxylase) . Oligonucleotide primers specific for the alkB gene of strain P . putida (GPo1 ) and for the alkB1 and alkB2 genes of P . aeruginosa strain PAO1 allowed amplification from the P . aeruginosa isolates of fragments similar to alkB1 and alkB2 genes of strain PAO1 . Only 3 strains carried an alkB gene very similar to that of strain GPo1, and these strains were the same ones that could utilise C6 to C10 n-alkanes.

Curr Microbiol, 2002 Nov, 45(5), 350 - 4
Transcription levels of Pseudomonas aeruginosa exotoxin a gene and severity of symptoms in patients with otitis externa; Matar GM et al.; Polymerase chain reaction (PCR)-based detection and transcription of the gene encoding a potent virulence factor, the exotoxin A, were done on 32 isolates of Pseudomonas aeruginosa belonging to 23 genotypes . These isolates were obtained from 22 patients who were admitted to the emergency room in a medical center during a 5-month period with the diagnosis of either unilateral or bilateral otitis externa . Patients showed symptoms that ranged from mild to severe . PCR amplification of a 396-bp fragment of the gene encoding the exotoxin A was done on extracted DNA . Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was performed on extracted RNA to detect exotoxin A gene mRNA transcripts . Quantitation of RT-PCR amplicons from P . aeruginosa isolates associated with mild and severe symptoms was determined by end-point titration of c-DNA and scanning of amplicons with the Storm Gel and Blot Imaging System . Data have shown that all of the 32 isolates of P . aeruginosa carry the exotoxin A gene, and all isolates with the exception of two had the exotoxin A transcription demonstrated by the production of a 396-bp amplicon from RT-PCR-amplified RNA . The remaining two isolates amplified fragments that were slightly smaller than the expected size . Additional studies are needed to characterize these two mRNA transcripts . Transcription levels of exotoxin A gene associated with severe symptoms were significantly more elevated than those associated with mild to moderate symptoms . Studies are under way to determine expression of P . aeruginosa exotoxin A by detecting quantitatively levels of the translated exotoxin A protein produced by isolates associated with severe and mild to moderate symptoms.

J Altern Complement Med, 2002 Aug, 8(4), 459 - 66
Gerimax ginseng regulates both humoral and cellular immunity during chronic Pseudomonas aeruginosa lung infection; Song Z et al.; BACKGROUND: Chronic lung infection among patients with cystic fibrosis (CF), diffused panbronchiolitis, and chronic obstructive bronchiecteisis is often because of Pseudomonas aeruginosa . High morbidity and mortality in patients with CF are because of P . aeuruginosa that undergoes genotypic and phenotypic changes during prolonged stay in the lung resulting in increased antibiotic resistance, necessitating a search for alternative or supplement drugs . OBJECTIVE: In this study we compared the therapeutical effect of Gerimax (Dansk Droge A/S, Ishoj, Denmark) ginseng with placebo control by using a rat model of chronic P . aeruginosa lung infection mimicking that in patients with CF . METHODS AND INTERVENTIONS: The animals were challenged intratracheally with the prototypic P . aeruginosa PAO1 in alginate beads (1 x 10(9) colony-forming units per milliliter {CFU/mL}) followed by subcutaneous injection of ginseng extract (150 mg/kg body weight once per day) and examined on days 7 and 21 . RESULTS: The day 7 analyses show that ginseng treatment resulted in lowering serum immunoglobulin M (IgM) and lung interleukin-4 (IL-4) levels compared to the control group . On day 21, higher lung IgA, upregulated serum IgG2a, stronger lung responses of interferon-gamma, IL-4, and tumor necrosis factor-alpha with milder lung pathology and enhanced lung bacteriology were detected in the ginseng-treated group when compared to those of the control group . CONCLUSION: These results suggest that the Gerimax ginseng treatment can modulate the immune system in favor of clearing the infection with P . aeruginosa in the lungs of rats . Thus, ginseng might be a promising alternative supplement for the treatment of chronic P . aeruginosa lung infection in patients with CF.

Chest, 2002 Sep, 122(3), 930 - 4
Evaluation of bronchial constriction in children with cystic fibrosis after inhaling two different preparations of tobramycin; Alothman GA et al.; OBJECTIVES: This randomized, double-blind, cross-over study evaluated the risk of bronchoconstriction with two preparations of inhaled tobramycin in children with cystic fibrosis (CF) infected with Pseudomonas aeruginosa with and without airway hyperreactivity . DESIGN: Of 19 children with CF (age range, 7 to 16 years) with mild-to-moderate pulmonary disease, 10 children were at high risk (HR) for bronchospasm (family history of asthma and previous response to bronchodilators) and 9 children were at low risk (LR) for bronchospasm (no family history of asthma or previous response to bronchodilators) . Two solutions of tobramycin were administered: (1) 80 mg in a 2-mL vial diluted with 2 mL of saline solution containing the preservatives phenol and bisulfites (IV preparation); and (2) 300 mg in a preservative-free preparation in a 5-mL solution . Following a bronchodilator-free period of 12 h, the patients inhaled either one or the other preparation in random order on two different occasions, 2 weeks apart . RESULTS: Prechallenge and postchallenge results for the LR group showed a percentage of fall in FEV(1) (DeltaFEV(1)) of 12 +/- 9% (mean +/- SD) for the IV preparation, compared to 4 +/- 5% for the preservative-free preparation (p = 0.046) . An DeltaFEV(1) of > 10% was seen in six of nine patients for the IV preparation and in one of nine patients for preservative-free preparation . For the HR group, the DeltaFEV(1) was 17 +/- 13% for the IV-preparation group, compared to 16 +/- 12% for the preservative-free group (p = 0.4) . In this group, equal numbers of patients (8 of 10 patients) had an DeltaFEV(1) > 10% after inhaling each preparation . The largest DeltaFEV(1) was 44% (HR group with the preservative-free preparation that forced the early termination of inhalation) . CONCLUSIONS: Both preparations caused significant bronchoconstriction in the HR group, and the preservative-containing IV preparation caused more bronchospasm in LR group than the preservative-free solution . Heightened airway reactivity in children with CF places them at risk of bronchospasm from inhalation therapy.

Microb Pathog, 2002 Sep, 33(3), 135 - 43
Mutation of csk, encoding the C-terminal Src kinase, reduces Pseudomonas aeruginosa internalization by mammalian cells and enhances bacterial cytotoxicity; Evans DJ et al.; Clinical isolates of Pseudomonas aeruginosa are either invasive or cytotoxic towards mammalian epithelial cells, endothelial cells, and macrophages . Invasion requires host cell actin cytoskeleton function, and ExsA-regulated proteins of P . aeruginosa that inhibit invasion (ExoS and ExoT) can disrupt the cytoskeleton . Another ExsA regulated protein, ExoU, is involved in the cytotoxic activity of cytotoxic strains . Src-family kinases are thought to participate in the regulation of cytoskeleton function . Recent studies have suggested that Src-family tyrosine kinases, p60-Src and p59-Fyn, are activated during P . aeruginosa invasion . Using fibroblasts homozygous for mutation of csk (-/-), we tested the hypothesis that mutation of csk, encoding a negative regulator of Src-family tyrosine kinases, would be important in P . aeruginosa invasion and cytotoxicity . Mutation of csk was found to reduce invasion by approximately 8-fold, without reducing bacterial adherence to cells (P=0.0001) . Conversely, csk (-/-) cells were approximately 5-fold more susceptible to ExoU-dependent cytotoxicity (P=0.024), which was accompanied by a small increase in ExsA-regulated adherence . ExoT-dependent invasion inhibitory activity of cytotoxic P . aeruginosa was attenuated in csk (-/-) cells as compared to normal fibroblasts . These data show that fibroblasts, like epithelial cells, are susceptible to P . aeruginosa invasion and cytotoxicity . They also show a role for Csk in P . aeruginosa invasion, while providing further evidence that actin cytoskeleton disruption contributes to ExsA-regulated P . aeruginosa cytotoxicity and invasion inhibition.

Microb Pathog, 2002 Sep, 33(3), 109 - 14
Pseudomonas aeruginosa outer membrane protein F is an adhesin in bacterial binding to lung epithelial cells in culture; Azghani AO et al.; Adherence to host cells is a crucial step by which bacteria initiate an infection but the bacterial determinants of the process are, as yet, poorly understood . In an effort to identify bacterial adhesins responsible for Pseudomonas aeruginosa binding to host cells, we identified porin F (OprF) from the outer membrane of P . aeruginosa as adhesin for human alveolar epithelial (A549) cells . Bacterial adhesion assays with (35)S-labeled wild type P . aeruginosa and its isogenic mutant strain lacking OprF showed that the mutant strain binds 43% less than the wild type to A549 cells (P<0.01) . In addition, bacterial binding is significantly reduced (P<0.01) when either A549 cells were pretreated with purified OprF or if bacteria were pre-incubated with a monoclonal antibody to OprF . Finally, ligand binding experiments in which purified OprF protein was added to A549 monolayers showed saturable binding . These data indicate that OprF contributes to bacterial adherence to A549 epithelial cells and could facilitate Pseudomonas interactions with the epithelium, including colonization of the airway epithelium or the initiation of pulmonary infection.

BMC Urol . 2002 Sep 10;2(1):9.
How should an infected perinephric haematoma be drained in a tetraplegic patient with baclofen pump implanted in the abdominal wall? - A case report; Vaidyanathan S et al.; BACKGROUND: We present a case to illustrate controversies in percutaneous drainage of infected, perinephric haematoma in a tetraplegic patient, who had implantation of baclofen pump in anterior abdominal wall on the same side as perinephric haematoma . CASE PRESENTATION: A 56-year-old male with C-4 tetraplegia had undergone implantation of programmable pump in the anterior abdominal wall for intrathecal infusion of baclofen to control spasticity . He developed perinephric haematoma while he was taking warfarin as prophylactic for deep vein thrombosis . Perinephric haematoma became infected with a resistant strain of Pseudomonas aeruginosa, and required percutaneous drainage . Positioning this patient on his abdomen without anaesthesia, for insertion of a catheter from behind, was not a realistic option . Administration of general anaesthesia in this patient in the radiology department would have been hazardous . RESULTS AND CONCLUSION: Percutaneous drainage was carried out by anterior approach under propofol sedation . The site of entry of percutaneous catheter was close to cephalic end of baclofen pump . By carrying out drainage from anterior approach, and by keeping this catheter for ten weeks, we took a risk of causing infection of the baclofen pump site, and baclofen pump with a resistant strain of Pseudomonas aeruginosa . The alternative method would have been to anaesthetise the patient and position him prone for percutaneous drainage of perinephric collection from behind . This would have ensured that the drainage track was far away from the baclofen pump with minimal risk of infection of baclofen pump, but at the cost of incurring respiratory complications in a tetraplegic subject.

J Am Anim Hosp Assoc, 2002 Sep-Oct, 38(5), 407 - 13
Frequency of isolation and antimicrobial susceptibility patterns of Staphylococcus intermedius and Pseudomonas aeruginosa isolates from canine skin and ear samples over a 6-year period (1992-1997); Petersen AD et al.; Staphylococcus intermedius (S . intermedius) was isolated from 88.6% and 49.4% of skin and ear samples, respectively, during the years 1992 through 1997, and frequency of isolation remained unchanged . More than 95% of all S . intermedius isolates were susceptible to cephalothin and oxacillin, providing support for empirical treatment of canine skin and ear infections with cephalexin . Pseudomonas aeruginosa (P . aeruginosa) was isolated from 7.5% and 27.8% of skin and ear samples, respectively . The frequency of isolation from skin samples increased over the study period . Because of multidrug-resistant profiles for P . aeruginosa isolates, especially for ear isolates, empirical treatment of P . aeruginosa infections is not advisable.

Hunan Yi Ke Da Xue Xue Bao, 2000 Aug 28, 25(4), 388 - 90
{Clinical study on nosocomial infection in patients with burns}; Yang XH et al.; OBJECTIVE: To study clinical characters and related factors of nosocomial infection in patients with burns . METHODS: To study 782 cases of burns as a group hospitalized in our department for more than 48 h, from January, 1993 to December, 1996 . Prospective and retrospective investigation of infection locations, infection rates, pathogen and drug sensibility was carried out . RESULTS: The infection most often found in blood and wound, less often in respiratory system . Nosocomial infection rates in patients with burns were closely related to the severe degree, diagnosis and therapy of these patients and hospital surroundings . Infection bacteria species: Gram-positive bacterium accounted for 43.82%, and mainly were staphylococcus aureus and anaerobic peptococcus . Gram-negative bacterium accounted for 52.81%, of which pseudomonas aeruginosa and nitrate-negative bacillus constituted the majority . CONCLUSION: According to these characters and related factors of nosocomial infection in patients with burns, effective measures of preventing and controlling infection should be taken, so that the incidence of nosocomial infection in these patients will drop.

Chemotherapy, 2002 Sep, 48(4), 182 - 8
Postantibiotic effect of meropenem and ciprofloxacin in the presence of 5-fluorouracil; Nyhlen A et al.; BACKGROUND: The postantibiotic effect (PAE) of meropenem and ciprofloxacin was studied in the presence of the antineoplastic agent 5-fluorouracil (5-FU) . The purpose of the study was to investigate whether the PAEs of the combinations differed from the PAEs of the antibiotics alone . METHODS: The PAEs of the combinations of 5-FU plus meropenem or ciprofloxacin were determined with viable counts against four reference strains of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli and two clinical isolates of S . epidermidis . The results were compared with the PAEs of the antibiotics drugs and 5-FU alone . The gram-positive strains were tested for slime production, both alone and in the presence of 5-FU . RESULTS: Against two of the three tested strains of S . epidermidis, the combination of ciprofloxacin and 5-FU gave a synergistic prolongation of the PAE in comparison with the PAEs induced by the drugs alone . The combinations showed indifference against the other bacteria . The combination of meropenem and 5-FU had a synergistic PAE against one of the three tested strains of S . epidermidis and an additive effect against E . coli but showed indifference against the rest of the strains . CONCLUSIONS: The presence of 5-FU did not influence the PAEs of the antibiotics against most of the tested strains, but caused a synergistic prolongation of the PAEs induced by ciprofloxacin and meropenem against some of the tested strains of S . epidermidis . 5-FU inhibited slime production in the same S . epidermidis strains, which might have contributed to the longer PAE .

Chemotherapy, 2002 Sep, 48(4), 164 - 7
Comparative study of antimicrobial resistance of Pseudomonas aeruginosa strains isolated from patients of Caracas and Asunción in a 4-year-period; Rodriguez AJ et al.; The antimicrobial resistance of Pseudomonas aeruginosa to beta-lactams, aminoglycosides and ciprofloxacin was investigated in two Latin American hospitals, one in Venezuela and the other in Paraguay . The resistance of P . aeruginosa was investigated in 1,481 clinically isolated strains, 988 from Asuncion and 493 from Caracas, collected between 1996 and 1999 . Susceptibility was assessed by the disk diffusion method according to the National Committee for Clinical Laboratory Standards . 11.4% were resistant to cefoperazone, 11.4% to ceftazidime, 12.8% to piperacillin, 13.6% to amikacin, 18.2% to gentamicin, 11.1% to ciprofloxacin, and 6.7% to imipenem . There were significant differences in resistance patterns between isolates from Asuncion and Caracas . Resistance was higher in Caracas . Despite similar antibiotic usage policies and other measures, differences in the resistance patterns of P . aeruginosa are evident in this study . The clinical and therapeutic implications of this resistance suggest the need to maintain surveillance in local settings, especially in developing countries such as Venezuela and Paraguay .

J Bacteriol, 2002 Oct, 184(19), 5251 - 60
FleQ, the major flagellar gene regulator in Pseudomonas aeruginosa, binds to enhancer sites located either upstream or atypically downstream of the RpoN binding site; Jyot J et al.; In Pseudomonas aeruginosa, flagellar genes are regulated in a cascade headed by FleQ, an NtrC/NifA-type activator . FleQ and RpoN positively regulate expression of flhA, fliE, fliL, and fleSR genes, among others . Direct interaction of FleQ with flhA, fliE, fliL, and fleSR promoters was demonstrated by gel shift assay, along with experiments to conclusively determine the specificity of its binding . DNase I footprinting was performed to determine the FleQ binding sites on flhA, fliE, fliL, and fleSR promoters . No sequence conservation among these binding sites was observed . Primer extension analysis revealed the transcription start sites (TSSs) to be localized above the FleQ binding sites in flhA, fliE, and fliL promoters . Analysis of the above data revealed FleQ binding to be in the leader sequence of these promoters, whereas FleQ binding was 67 bp upstream of the TSS in the fleSR promoter . Mutagenesis of the FleQ binding site in the flhA promoter confirmed its functionality in vivo . Deletion of the flhA promoter upstream of the RNA polymerase binding site did not result in a significant loss of promoter activity . These results point to two modes of regulation by an NtrC-type regulator in the flagellar hierarchy in P . aeruginosa, the first being the typical model of activation from a distance via looping in the fleSR promoter and the second involving flhA, fliE, and fliL promoters, where FleQ binds in the downstream vicinity of the promoter and activates transcription without looping.

J Bacteriol, 2002 Oct, 184(19), 5240 - 50
fleQ, the gene encoding the major flagellar regulator of Pseudomonas aeruginosa, is sigma70 dependent and is downregulated by Vfr, a homolog of Escherichia coli cyclic AMP receptor protein; Dasgupta N et al.; The flagellar transcriptional regulator FleQ appears to be the highest-level regulator in the hierarchical regulatory cascade of flagellar biogenesis in Pseudomonas aeruginosa . Except for the posttranslational downregulation of FleQ activity by FleN, an antiactivator, not much is known about the regulation of the fleQ gene or its gene product . Some FleQ homologs in other bacterial species either are positively regulated by another regulator (e.g., CtrA, the master regulator regulating FlbD in Caulobacter crescentus) or are expressed from a sigma70-dependent promoter (e.g., FlgR of Helicobacter pylori) . In this study we demonstrated that Vfr, an Escherichia coli CRP homolog known to function as an activator for various genes, including lasR, regA, and toxA, in P . aeruginosa, is capable of repressing fleQ transcription by binding to its consensus sequence in the fleQ promoter . In a DNase I footprint assay, purified Vfr protected the sequence 5'-AATTGACTAATCGTTCACATTTG-3' . When this putative Vfr binding site in the fleQ promoter was mutated, Vfr was unable to bind the fleQ promoter fragment and did not repress fleQ transcription effectively . Primer extension analysis of the fleQ transcript revealed two transcriptional start sites, t1 and t2, that map within the Vfr binding site . A putative -10 region (TAAAAT) for the t2 transcript, with a five-of-six match with the E . coli sigma70 binding consensus, overlaps with one end of the Vfr binding site . A 4-bp mutation and an 8-bp mutation in this -10 region markedly reduced the activity of the fleQ promoter . The same mutations led to the disappearance of the 203-nucleotide fleQ transcript in an in vitro transcription assay . Vfr probably represses fleQ transcription by binding to the Vfr binding site in the fleQ promoter and preventing the sigma factor from binding to the -10 region to initiate transcription.

Bioorg Med Chem, 2002 Nov, 10(11), 3489 - 98
Design, synthesis, and biological evaluation of a series of beta-lactam-based prodrugs; Hakimelahi GH et al.; By use of pro-dual-drug concept the synthesis of 6-beta-{(R)-2-(clavaminio-9-N-yl)-2-(4-hydroxyphenylacetamido)}penicillanic acid (10), 6-beta-{(R)-2-(amino)-2-(4-(clavulano-9-O-yl)phenylacetamido)}penicillanic acid (13), (Z)-4-{2-(amoxycillin-4-O-yl)ethylidene}-2-(clavulano-9-O-yl)-3-methoxy-Delta(alpha,beta)-butenolide (19), and 3-{(amoxicillin-4-O-yl)methyl}-7-(phenoxyacetamido)-(1-oxo)-3-cephem-4-carboxylic acid (23) was accomplished . Unlike penicillin G, ampicillin, or amoxicillin, these four heretofore undescribed compounds 10, 13, 19, and 23 showed notable activity against beta-lactamase (betaL) producing microorganisms, Staphylococcus aureus A9606, S . aureus A15091, S . aureus A20309, S . aureus 95, Escherichia coli A9675, E . coli A21223, E . coli 27C7, Pseudomonas aeruginosa 18S-H, and Klebsiella pneumoniae A20634 TEM . In comparison with amoxicillin (9), alpha-amino-substituted compound 10 and butenolide derivative 19 showed a broadened spectrum of antibacterial activity; yet they were found to be less active than 13 and 23 . Like clavulanic acid (7) or cephalosporin-1-oxide (21), the newly synthesized compounds 10, 13, 15, 16, 19, or 23 functioned as potent inhibitors of various bacterial betaLs.

J Pept Sci, 2002 Aug, 8(8), 431 - 7
Antimicrobial activity of short arginine- and tryptophan-rich peptides; Strom MB et al.; Highly antimicrobial active arginine- and tryptophan-rich peptides were synthesized ranging in size from 11 to five amino acid residues in order to elucidate the main structural requirement for such short antimicrobial peptides . The amino acid sequences of the peptides were based on previous studies of longer bovine and murine lactoferricin derivatives . Most of the peptides showed strong inhibitory action against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive bacterium Staphylococcus aureus . For the most active derivatives, the minimal inhibitory concentration values observed for the Gram-negative bacteria were 5 microg/ml (3.5 microM), whereas it was 2.5 microg/ml (1.5 microM) for the Gram-positive bacterium . It was essential for the antimicrobial activity that the peptides contained a minimum of three tryptophan and three arginine residues, and carried a free N-terminal amino group and an amidated C-terminal end . Furthermore, a minimum sequence size of seven amino acid residues was required for a high antimicrobial activity against Pseudomonas aeruginosa . The insertion of additional arginine and tryptophan residues into the peptides resulted only in small variations in the antimicrobial activity, whereas replacement of a tryptophan residue with tyrosine in the hepta- and hexapeptides resulted in reduced antimicrobial activity, especially against the Gram-negative bacteria . The peptides were non-haemolytic, making them highly potent as prospective antibiotic agents.

Kansenshogaku Zasshi, 2002 Jul, 76(7), 576 - 80
{A case of Pseudomans aeruginosa pneumonia complicated with multiple pustular skin lesions}; Miyajima Y et al.; Pseudomonas aeruginosa is a common causative agent of septicemia in compromised host and the entry site of organism is most commonly the respiratory and genitourinary tract . P . aeruginosa septicemia is often associated with vesicular or pustular skin lesions, subcutaneous nodules, deep abscess, cellulites and bullae . We report a case of P . aeruginosa pneumonia with multiple pustular skin lesions on the chest and leg . A 77-year-old male was admitted to our hospital complaining of fever, productive cough and eruptions . Laboratory findings revealed a leucocytosis (14,830/microliter) and an elevated CRP (21.72 mg/dl) . The chest radiograph and computed tomography revealed a fluid level in preexisting bullae and a consolidation shadow with multiple cavities in the right upper lobe and nodular shadow with cavity in the left lower lobe . P . aeruginosa strain was isolated from the bronchial lavage and pustule . Blood cultures were negative . Skin biopsy specimens showed histologically a dense infiltrate of neutrophils in the horny cell layer . He was diagnosed as Pseudomonas aeruginosa pneumonia complicated with multiple pustular skin lesions . He was treated with antimicrobial agents for 24 days and his clinical condition improved.

Mol Microbiol, 2002 Sep, 45(5), 1277 - 87
GeneChip expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes; Ochsner UA et al.; Upon iron restriction, the opportunistic pathogen Pseudomonas aeruginosa produces various virulence factors, including siderophores, exotoxin, proteases and haemolysin . The ferric uptake regulator (Fur) plays a central role in this response and also controls other regulatory genes, such as pvdS, which encodes an alternative sigma factor . This circuit leads to a hierarchical cascade of direct and indirect iron regulation . We used the GeneChip to analyse the global gene expression profiles in response to iron . In iron-starved cells,the expression of 118 genes was increased at least fivefold compared with that in iron-replete cells, whereas the expression of 87 genes was decreased at least fivefold . The GeneChip data correlated well with results obtained using individual lacZ gene fusions . Strong iron regulation was observed for previously identified genes involved in biosynthesis or uptake of the siderophores pyoverdine and pyochelin, utilization of heterologous siderophores and haem and ferrous iron transport . A low-iron milieu led to increased expression of the genes encoding TonB, alkaline protease,PrpL protease, exotoxin A, as well as fumarase C, Mn-dependent superoxide dismutase SodA, a ferredoxin and ferredoxin reductase and several oxidoreductases and dehydrogenases . Iron-controlled regulatory genes included seven alternative sigma factors and five other transcriptional regulators . Roughly 20% of the iron-regulated genes encoded proteins of unknown function and lacked any conclusive homologies . Under low-iron conditions, expression of 26 genes or operons was reduced in a DeltapvdS mutant compared with wild type, including numerous novel pyoverdine biosynthetic genes . The GeneChip proved to be a very useful tool for rapid gene expression analysis and identification of novel genes controlled by Fur or PvdS.

Mol Microbiol, 2002 Sep, 45(5), 1177 - 90
Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas; Visca P et al.; A variety of bacterial species secrete and take up chelating compounds that enable acquisition of iron (siderophores) . It has become clear that a common feature in regulation of different iron acquisition systems is the involvement of alternative sigma factor proteins of the extracytoplasmic function (ECF) family . Two of these proteins, PvdS from Pseudomonas aeruginosa and FecI from Escherichia coli K-12, have been studied extensively . PvdS directs transcription of genes required for the biosynthesis of a siderophore, pyoverdine, and FecI causes expression of genes for uptake of ferric citrate . FecI forms part of a signalling system that responds to the presence of ferric citrate . Here, we review recent advances in understanding of PvdS and of the Fec signalling system . PvdS and FecI are part of a distinct subfamily of ECF sigma factors involved in iron acquisition and hence named the iron-starvation sigmas . Analysis of microbial genome sequences shows that Fec-like signalling systems are present in a wide range of species and many such systems may be present in a single species . The availability of tools for large-scale genome analysis is likely to lead to rapid advances in our understanding of this expanding family of proteins.

Pediatr Pulmonol, 2002 Oct, 34(4), 257 - 61
Transmission of colistin-resistant Pseudomonas aeruginosa between patients attending a pediatric cystic fibrosis center; Denton M et al.; We report on an outbreak of colistin-resistant Pseudomonas aeruginosa (CRPA) that occurred in a United Kingdom pediatric cystic fibrosis (CF) unit and involved six children over a period of 5 years . All CRPA-positive children had received aerosolized colistin therapy before first isolation of resistant organisms (mean duration, 3.1 years) . Four of the 6 had also received courses of intravenous colistin in the year before the first isolation of CRPA . No impact of CRPA acquisition on respiratory function, clinical condition, or radiological parameters could be demonstrated . Four of the 6 children carried isolates of CRPA indistinguishable on genotyping . Two of these 4 children were sisters . The other 2 were on the same ward together at time of first isolation, and subsequently shared overlapping admissions with one of the sisters.While there is no conclusive evidence for the route of transmission, the frequency of overlapping in-patient admissions between 3 of these patients is suggestive of patient-to-patient transfer in the nosocomial setting.CF clinicians should be aware that colistin resistance can occur in P . aeruginosa, and some of these strains are capable of spread within CF units .

Pediatr Pulmonol, 2002 Sep, 34(3), 232 - 6
Early infection and progression of cystic fibrosis lung disease; Koch C; Chronic Pseudomonas aeruginosa infection is the major limitation in overall survival of patients with cystic fibrosis, and elective bronchoscopy shows that infection with this organism starts at a very early age . Early infection with nonmucoid P . aeruginosa gradually develops into chronic infection, characterized by the presence of microcolonies of alginate-producing (mucoid) P . aeruginosa in the bronchial tree . Chronic infection cannot be cured, but aggressive antimicrobial treatment will prolong life expectancy and decrease morbidity . It has, however, over the last decade become evident that early antimicrobial treatment of the initial infection with nonmucoid strains can prevent or at least postpone transition into chronic (mucoid) infection . This treatment strategy is likely to dramatically improve the prognosis of patients with cystic fibrosis in the immediate future .

Anal Chem, 2002 Aug 15, 74(16), 4290 - 3
Simultaneous multiple substrate tag detection with ESI-ion trap MS for in vivo bacterial enzyme activity profiling; Basile F et al.; A bacterial identification method in which multiple enzyme activities are measured simultaneously and in vivo with electrospray ionization-mass spectrometry (ESI-MS) is described . Whole-cell bacteria are immobilized onto a filter support and incubated with a mixture of substrates . Each substrate is chosen to measure a specific enzyme activity of a targeted bacterium and to produce a tag of unique molecular weight . After a predetermined incubation time, the solution is filtered, and the supernatant consisting of a mixture of released tags and unhydrolyzed substrates is directly analyzed, without chromatographic separation, by ESI-MS . Bacteria remain viable on the filter for further analyses . The method was tested by measuring the aminopeptidase activity of the bacteria Escherichia coli, Bacillus subtilis, Bacillus cereus, and Pseudomonas aeruginosa . The resulting aminopeptidase enzyme profiles allowed the differentiation between the four bacteria tested . The method is rapid, since a multiplex advantage is realized when assaying for multiple enzymes, and it is amenable to automation via a flow injection analysis setup.

Biochem Soc Trans, 2002 Aug, 30(4), 702 - 5
A new mechanism for membrane iron transport in Pseudomonas aeruginosa; Schalk IJ et al.; Various biochemical and biophysical studies have demonstrated the existence of a novel iron-uptake mechanism in Pseudomonas aeruginosa, different from that generally described for ferrichrome and ferric-enterobactin in Escherichia coli . This new iron-uptake mechanism involves all the proteins generally reported to be involved in the uptake of ferric-siderophore complexes in Gram-negative bacteria (i.e . the outer membrane receptor, periplasmic binding protein and ATP-binding-cassette transporter), but differs in the behaviour of the siderophore . One of the key features of this process is the binding of iron-free pyoverdin to the outer membrane receptor FpvA in conditions of iron deficiency.

Arch Intern Med, 2002 Sep 9, 162(16), 1849 - 58
Community-acquired pneumonia due to gram-negative bacteria and pseudomonas aeruginosa: incidence, risk, and prognosis; Arancibia F et al.; BACKGROUND: Initial empirical antimicrobial treatment of patients with community-acquired pneumonia (CAP) is based on expected microbial patterns . We determined the incidence of, prognosis of, and risk factors for CAP due to gram-negative bacteria (GNB), including Pseudomonas aeruginosa . METHODS: Consecutive patients with CAP hospitalized in our 1000-bed tertiary care university teaching hospital were studied prospectively . Independent risk factors for CAP due to GNB and for death were identified by means of stepwise logistic regression analysis . RESULTS: From January 1, 1997, until December 31, 1998, 559 hospitalized patients with CAP were included . Sixty patients (11%) had CAP due to GNB, including P aeruginosa in 39 (65%) . Probable aspiration (odds ratio {OR}, 2.3; 95% confidence interval {CI}, 1.02-5.2; P =.04), previous hospital admission (OR, 3.5; 95% CI, 1.7-7.1; P<.001), previous antimicrobial treatment (OR, 1.9; 95% CI, 1.01-3.7; P =.049), and the presence of pulmonary comorbidity (OR, 2.8; 95% CI, 1.5-5.5; P =.02) were independent predictors of GNB . In a subgroup analysis of P aeruginosa pneumonia, pulmonary comorbidity (OR, 5.8; 95% CI, 2.2-15.3; P<.001) and previous hospital admission (OR, 3.8; 95% CI, 1.8-8.3; P =.02) were predictive . Infection with GNB was independently associated with death (relative risk, 3.4; 95% CI, 1.6-7.4; P =.002) . CONCLUSIONS: In our setting, in every tenth patient with CAP, an etiology due to GNB has to be considered . Patients with probable aspiration, previous hospitalization or antimicrobial treatment, and pulmonary comorbidity are especially prone to GNB . These pathogens are also an independent risk factor for death in patients with CAP.

Eye, 2002 Sep, 16(5), 608 - 18
Incidence and risk factors for microbial keratitis in Hong Kong: comparison with Europe and North America; Lam DS et al.; PURPOSE: To establish the incidence, etiology and risk factors for microbial keratitis (MK) in Hong Kong . METHODS: Two hundred and twenty-three new cases of presumed MK were recruited over a period of 17 months and comprehensive microbiologic studies performed . A nested case-control study was pursued for patients wearing contact lenses (CLW) to determine risk factors for MK with regards to types of CLW and hygiene practice . RESULTS: Of the 223 patients recruited, 59 (26%) wore contact lenses . Corneal scrapes yielded positive cultures from 77 patients (35% overall, 56 non-CLW, 21 CLW) . Two hundred and six CLW volunteers were recruited to participate in the case-control study, of whom 135 were matched with 45 CLW patients . The annual incidence of MK was 0.63 per 10,000 population and 3.4 per 10,000 CLW with rates for daily, extended and rigid lens wear of 3.09, 9.30 and 0.44 per 10,000 CLW respectively . Pseudomonas aeruginosa was the dominant bacterial pathogen . Six cases of Acanthamoeba keratitis occurred, five in CLW (incidence 0.33 per 10,000 CLW) and one following corneal abrasion . Non-CLW developed MK at a peak age of 73, which is 10 years younger than expected for Scotland and USA . CONCLUSIONS: Previous ocular surface disease and trauma were the main risk factors for MK in Hong Kong . CLW appears at least as safe as that found in Scotland and the USA . Acanthamoeba keratitis was detected but with an incidence rate five times lower than Scotland . Factors predisposing hydrogel CLWs to MK, that were statistically significant, included overnight wear, poor hygiene and smoking.

J Immunol, 2002 Sep 1, 169(5), 2636 - 42
The Pseudomonas autoinducer N-(3-oxododecanoyl) homoserine lactone induces cyclooxygenase-2 and prostaglandin E2 production in human lung fibroblasts: implications for inflammation; Smith RS et al.; Pseudomonas aeruginosa causes lethal lung infections in immunocompromised individuals such as those with cystic fibrosis . The lethality of these infections is directly associated with inflammation and lung tissue destruction . P . aeruginosa produces several acylated homoserine lactones (AHL) that are important in the regulation of bacterial virulence factors . Little is known about the effects of AHLs on human cells . In this work we report that the AHL N-(3-oxododecanoyl) homoserine lactone (3O-C(12)-HSL) from P . aeruginosa induces cyclooxygenase (Cox)-2, a seminal proinflammatory enzyme . When primary normal human lung fibroblasts were exposed to 3O-C(12)-HSL, an 8-fold induction in mRNA and a 35-fold increase in protein for Cox-2 were observed . In contrast, there was no substantial change in the expression of Cox-1 . We also demonstrated that the induction of Cox-2 was regulated by 3O-C(12)-HSL activation of the transcription factor NF-kappaB . 3O-C(12)-HSL also stimulated an increase in the newly discovered inducible membrane-associated PGE synthase but had no effect on the expression of the cytosolic PGE synthase . We also demonstrate that 3O-C(12)-HSL stimulated the production of PGE(2) . PGE(2) is known to induce mucus secretion, vasodilation, and edema, and acts as an immunomodulatory lipid mediator . We propose that 3O-C(12)-HSL induction of Cox-2, membrane-associated PGE synthase, and PGE(2) likely contributes to the inflammation and lung pathology induced by P . aeruginosa infections in the lung . These studies further reinforce the concept that bacterial AHLs not only regulate bacterial virulence but also stimulate the activities of eukaryotic cells important for inflammation and immune defenses.

J Bacteriol, 2002 Sep, 184(18), 5036 - 44
The MexJK efflux pump of Pseudomonas aeruginosa requires OprM for antibiotic efflux but not for efflux of triclosan; Chuanchuen R et al.; Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible Pseudomonas aeruginosa strain were isolated . Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the mexJK operon . Expression of this operon is negatively regulated by the product of mexL, a gene located upstream of and transcribed divergently from mexJK . The triclosan-resistant mutant contained a single nucleotide change in mexL, which caused an amino acid change in the putative helix-turn-helix domain of MexL . The MexL protein belongs to the TetR family of repressor proteins . The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK . Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter . The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system . Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics.

Hosp Med, 2002 Jul, 63(7), 421 - 5
TOBI: reducing the impact of pseudomonal infection; Govan J; Pseudomonas aeruginosa infection is associated with impaired lung function and reduced life expectancy in cystic fibrosis patients . Tobramycin nebulizer solution (TOBI, Chiron Corporation Ltd, Hounslow) has been specifically formulated for use against P . aeruginosa infection in the lung.

Infect Control Hosp Epidemiol, 2002 Aug, 23(8), 441 - 6
An outbreak of multidrug-resistant Pseudomonas aeruginosa associated with increased risk of patient death in an intensive care unit; Bukholm G et al.; OBJECTIVE: To investigate an outbreak of multidrug-resistant Pseudomonas aeruginosa in an intensive care unit (ICU) . DESIGN: Epidemiologic investigation, environmental assessment, and ambidirectional cohort study . SETTING: A secondary-care university hospital with a 10-bed ICU . PATIENTS: All patients admitted to the ICU receiving ventilator treatment from December 1, 1999, to September 1, 2000 . RESULTS: An outbreak in an ICU with multidrug-resistant isolates of P aeruginosa belonging to one amplified fragment-length polymorphism (AFLP)-defined genetic cluster was identified, characterized, and cleared . Molecular typing of bacterial isolates with AFLP made it possible to identify the outbreak and make rational decisions during the outbreak period . The outbreak included 19 patients during the study period . Infection with bacterial isolates belonging to the AFLP cluster was associated with reduced survival (odds ratio, 5.26; 95% confidence interval, 1.14 to 24.26) . Enhanced barrier and hygiene precautions, cohorting of patients, and altered antibiotic policy were not sufficient to eliminate the outbreak . At the end of the study period (in July), there was a change in the outbreak pattern from long (December to June) to short (July) incubation times before colonization and from primarily tracheal colonization (December to June) to primarily gastric or enteral July) colonization . In this period, the bacterium was also isolated from water taps . CONCLUSION: Complete elimination of the outbreak was achieved after weekly pasteurization of the water taps of the ICU and use of sterile water as a solvent in the gastric tubes.

Med Dosw Mikrobiol, 2002, 54(1), 61 - 6
{The effect of culture conditions on hydrophobic properties of Pseudomonas aeruginosa}; Wolska K et al.; The cell surface hydrophobicity (CSH) plays an important role in a adhesion of bacteria on solid surfaces . CSH of 62 Pseudomonas aeruginosa strains isolated from humans and different animals was assessed using the ammonium sulfate salt aggregation test . Bacteria were grown for 24 h and 48 h at a room temperature (22 degrees C) and 37 degrees C on enrichment broth and agar (Biomed) and tryptic soy agar (Difco) . The hydrophobic properties of the Pseudomonas aeruginosa strains were depended on the temperature, time of the culture of bacteria and the kind of media . CSH properties were most frequently expressed when the analyzed strains were cultured in enrichment broth . In a such conditions Pseudomonas aeruginosa strains were more hydrophobic when grown at 22 degrees C (94% after 24 h and 87% after 48%) than those at 37 degrees C (72% after 24 h and 71% after 48 h) . Among strains cultured in tryptic soy agar at 37 degrees C, 48% after 24 h and 75% after 48 h were autoaggregating, representing very strong hydrophobic properties.

Br J Ophthalmol, 2002 Sep, 86(9), 975 - 7
The use of surgical facemasks during cataract surgery: is it necessary?
Alwitry A, Jackson E, Chen H, Holden R.
AIM: To assess whether facemask utilisation by the surgeon during cataract surgery has any effect on the bacterial load falling onto the operative site . METHOD: Prospective randomised masked study . Consent was obtained from 221 patients . Cases were randomised to wearing a new mask or not wearing any mask throughout the procedure . Blood agar settle plates were placed adjacent to the patient's head in the operative field . Duration of procedure was noted . Plates were incubated and read at 48 hours . Colony forming bacteria were counted and identified . RESULTS: There were significantly fewer organisms cultured when the surgeon used a facemask (p=0.0006) . The majority of organisms were Staphylococcus epidermidis, Bacillus spp, and Diphtheroid spp; however Staphylococcus aureus and Pseudomonas aeruginosa were cultured on several occasions . There were no cases of infective complication . CONCLUSIONS: The main purpose of an operating mask is to prevent bacteria falling on to the operative site from the surgeon's oropharynx or nasopharynx with the concomitant theoretical risk of infective complication . Operating masks were shown to have a significant effect on the volume of bacterial organisms falling to the operative site; however, whether this is clinically significant is unknown.

J Pharmacol Exp Ther, 2002 Sep, 302(3), 1151 - 7
Fluticasone propionate inhibits lipopolysaccharide-induced proinflammatory response in human cystic fibrosis airway grafts; Escotte S et al.; Airway inflammation, one of the major factors leading to lung damage in cystic fibrosis (CF) patients, is associated with an abnormal increase in proinflammatory cytokines . In this work, we demonstrate the increased release of the proinflammatory cytokines after lipopolysaccharide (LPS) stimulation: human interleukin (hIL)-8 in CF and non-CF airway xenografts, and hIL-6 and human growth-related oncogene-alpha (hGRO-alpha), which could be only analyzed in non-CF xenografts . Under basal conditions, we observed that hIL-8 was higher in CF xenografts compared with non-CF . We also report the anti-inflammatory effect of a glucocorticoid, fluticasone propionate (FP), on CF airway epithelium using a humanized model of airway inflammation developed in nude mice . In CF and non-CF tracheal xenografts, airway inflammation was induced by inoculating Pseudomonas aeruginosa LPS (4 h; 1 microg/ml) in the lumen of the xenografts . FP pretreatment (2 h; 10(-8) M) followed by P . aeruginosa LPS stimulation induced a significant reduction of LPS-induced hIL-8 release in airway liquid collected from CF and non-CF tracheal xenografts (85 and 80%, respectively) . In non-CF tracheal xenografts, FP treatment before LPS stimulation induced a significant decrease in hIL-6 and hGRO-alpha . From these data, we suggest that FP exerts anti-inflammatory properties that may be appropriate to CF therapy, at an early stage of the disease . In addition, these results demonstrate that the humanized airway model of inflammation provides a relevant tool for analyzing the effects of anti-inflammatory drugs in different diseases in which airway inflammation is implicated.

Antimicrob Agents Chemother, 2002 Sep, 46(9), 3031 - 4
Prospective survey of beta-lactamases produced by ceftazidime- resistant Pseudomonas aeruginosa isolated in a French hospital in 2000; De Champs C et al.; In 2000, at the Universite d'Auvergne teaching hospital in Clermont-Ferrand, France, 44 (6.2%) strains of Pseudomonas aeruginosa were found to be resistant to ceftazidime . After genotyping, 34 strains were selected . Nine had an additional beta-lactamase: OXA-21 (n = 6), PSE-1 (CARB-2) (n = 2), or PER-1 (n = 1) . Ceftazidime resistance was related solely to the overproduction of the cephalosporinase in 30 strains . Sequencing of five bla(AmpC) genes encoding cephalosporinases with different pIs showed 99% identity with the ampC gene of P . aeruginosa PAO1.

Antimicrob Agents Chemother, 2002 Sep, 46(9), 2920 - 5
Consumption of imipenem correlates with beta-lactam resistance in Pseudomonas aeruginosa; Lepper PM et al.; It is generally assumed that the antibiotic prescription policy of a hospital has a significant impact on bacterial resistance rates; however, few studies are available to support this concept with valid statistical data . During a 3-year period from 1997 to 2000, we monitored the consumption of beta-lactam and other antibiotics with known activity against Pseudomonas aeruginosa in a 600-bed community hospital . Monthly isolations of P . aeruginosa were assessed, and resistance rates were recorded . Partial correlation coefficients between consumption and resistance rates were determined, taking into account possible associations with other variables such as seasonal effects and transfers from other hospitals . A total of 30 +/- 7 novel P . aeruginosa strains per month were isolated without epidemic clustering . Prescriptions of imipenem varied significantly during the study period, while prescriptions of other antipseudomonal agents were stable, with the exception of an increase in piperacillin-tazobactam prescriptions . Rates of resistance of P . aeruginosa to the antimicrobial agents used showed a time course similar to figures for imipenem consumption . Monthly rates of resistance to imipenem (partial correlation coefficient {cc}, 0.63), piperacillin-tazobactam (cc, 0.57), and ceftazidime (cc, 0.56) were significantly associated with imipenem prescription rates in the same or the preceding month, while consumption of ceftazidime or piperacillin-tazobactam had no apparent association with resistance . Among the variables investigated, imipenem consumption was identified as the major factor associated with both carbapenem and beta-lactam resistance in endemic P . aeruginosa . Periods of extensive imipenem use were associated with significant increases in resistance . Our data support the concept that a written antibiotic policy which balances the use of various antibiotic classes may help to avoid disturbances of a hospital's microbial sensitivity patterns.

Lett Appl Microbiol, 2002, 35(3), 212 - 7
Zinc and glycerol enhance the production of nematicidal compounds in vitro and improve the biocontrol of Meloidogyne javanica in tomato by fluorescent pseudomonads; Siddiqui IA et al.; AIMS: To assess the effects of various carbon and mineral sources on the nematicidal potential of biocontrol inoculants of Pseudomonas aeruginosa IE-6S+ and Ps . fluorescens CHA0 under laboratory and glasshouse conditions . METHODS AND RESULTS: Culture filtrates of strains IE-6S+ and CHA0, cultured in nutrient yeast extract broth, caused substantial mortality of the juveniles of Meloidogyne javanica . The nematicidal activities of the culture filtrates were altered after amendment with various carbon and mineral sources . Soil amendment with zinc alone or in combination with glycerol improved the biocontrol efficacy against root-knot nematode, promoted tomato plant growth and enhanced bacterial rhizosphere and endophytic colonization . CONCLUSIONS: Appropriate quantities of glycerol and zinc alone or in combination enhance the nematicidal activity of Ps . aeruginosa and Ps . fluorescens . Glucose reduces the activity of these bacteria against nematodes . SIGNIFICANCE AND IMPACT OF THE STUDY: Minerals and carbon sources are appealing because they are easy and economical to provide during liquid fermentation of inoculants or as fertilizer amendments to improve the biocontrol activity of indigenous and introduced bacteria.

Br J Oral Maxillofac Surg, 2002 Apr, 40(2), 175 - 6
Bilateral eyelid necrosis as a complication of pseudomonal septicaemia; Dickenson AJ et al.; We present a case of bilateral eyelid necrosis as a result of Pseudomonas aeruginosa infection . This is a rare condition that occurs only in neutropenic patients . It may be unilateral or bilateral and requires aggressive management with correction of the neutrophil count, local debridement and systemic antibiotic therapy.

DNA Seq, 2002 Feb, 13(1), 67 - 74
Isolation and sequence analysis of the GlnKamtB1amtB2 gene cluster, encoding a PII homologue and two putative ammonium transporters, from Pseudomonas stutzeri A15; Vermeiren H et al.; By PCR, using primers based on heterologous amtB genes, an amtB sequence of Pseudomonas stutzeri A15 was amplified . This DNA fragment was used as a probe in Southern hybridisation experiments and resulted in the isolation and sequence analysis of a 6017 bp genomic fragment of P . stutzeri A15 containing glnKamtB1amtB2 . GlnK codes for a homologue of the nitrogen regulatory PII protein, amtB1 and amtB2 encode putative ammonium transporters . Whereas a glnKamtB gene cluster is common among bacteria, a tandem repeat of ammonium transporter genes has not been reported before . Apart from the presence of a second amtB gene, the gene organisation on this 6 kbp fragment is very similar to a particular region in the genome of Pseudomonas aeruginosa PAO1, relatively closely related to P . stutzeri . Furthermore, the amtB1 gene shows the highest similarity with P . aeruginosa amtB, whereas the amtB2 gene is more closely related to cyanobacterial amtB genes, which are reported to be monocistronically transcribed and not clustered with glnK homologues . Upstream of glnK, NtrC and RpoN recognition sites can be observed . In the intergenic region of glnKamtB1amtB2 no terminators nor extra promoter sequences were observed, indicating that glnKamtB1amtB2 is possibly transcribed as a nitrogen regulated operon.

Microbiology, 2002 Aug, 148(Pt 8), 2449 - 56
The ferric uptake regulator of Pseudomonas aeruginosa has no essential cysteine residues and does not contain a structural zinc ion; Lewin AC et al.; The ferric uptake regulator (Fur) of Pseudomonas aeruginosa was expressed in Escherichia coli in its native form and as a fusion to the maltose-binding protein (MBP) . Fur from the MBP fusion bound to MBP after proteolytic cleavage, and the two could only be separated by partial unfolding . The refolded protein was in the same conformation as native protein (as judged by circular dichroism and fluorescence spectroscopies) and was fully active in DNA-binding assays . As-prepared native Fur contained small amounts of Zn(2+) that were easily removed by treatment with EDTA, and apo-protein could be reconstituted with approximately one Zn(2+) ion per monomer . Thus, the P . aeruginosa Fur can probably accommodate a single Zn(2+) ion bound to the metal-sensing site . The single cysteine residue of P . aeruginosa Fur aligns with a cysteine in other members of the Fur family that is essential for activity of the E . coli protein, and is believed to provide one of the ligands to a structural Zn(2+) ion . This cysteine residue was shown to be dispensable for the in vivo activity of P . aeruginosa Fur, which is consistent with the suggestion that the P . aeruginosa protein does not contain a structural Zn(2+) ion . Members of the Fur family contain a highly conserved His-His-Asp-His motif . Alanine substitutions of residues in this motif showed His-87 and His-89 of P . aeruginosa Fur to be essential for activity, whilst His-86 and Asp-88 are partially dispensable.

Microbiology, 2002 Aug, 148(Pt 8), 2371 - 81
Characterization of a new efflux pump, MexGHI-OpmD, from Pseudomonas aeruginosa that confers resistance to vanadium; Aendekerk S et al.; Vanadium has an antibacterial activity against Pseudomonas aeruginosa, especially under conditions of iron limitation . Some degree of resistance to V is inducible by prior exposure to the metal . One mutant (VS1) with a higher sensitivity to V was obtained by transposon mutagenesis of P . aeruginosa PA 59.20, a clinical isolate . This mutant had an insertion in a non-coding region, upstream of a cluster of four genes . Three of them show similarities to genes corresponding to known P . aeruginosa antibiotic efflux systems, including an efflux protein, a membrane fusion protein and an outer-membrane porin . This cluster was named mexGHI-opmD . By allelic exchange, three mutants, ncr (for non-coding region), mexI and opmD were constructed in P . aeruginosa PAO1 . Next to V sensitivity, the ncr, mexI and opmD mutants also showed reduced production of elastase, rhamnolipids, pyocyanine, pyoverdine and had reduced swarming motility, phenotypes that are known to be regulated by quorum sensing . All wild-type phenotypes, including growth in the presence of V, were restored by complementation with the complete cluster . The production of N-acyl-homoserine lactones (AHLs) was detected using the Chromobacter violaceum bioassay . Total extracts from the three mutants failed to induce the production of violacein by C . violaceum, although AHLs were detected by TLC and C . violaceum overlay . Violacein production was restored by complementation with mexGHI-opmD . The opmD mutant grew very slowly in LB or CAA medium, indicating that OpmD has an important physiological function for the cell . In conclusion, it is believed that the MexGHI-OpmD pump is probably involved in AHL homeostasis in P . aeruginosa.

Saudi Med J, 2002 Jul, 23(7), 802 - 5
Effects of some plant extracts and antibiotics on Pseudomonas aeruginosa isolated from various burn cases; Al-Saimary IE et al.; OBJECTIVE: To determine the major groups of bacteria associated with burn infections, isolation of Pseudomonas aeruginosa from burn cases, and the testing of antibacterial activity of some plant extracts in comparison with the standard antibiotics . METHODS: A total of 92 burn cases, covered with tetracycline (40 cases) and non-covered with tetracycline (52 cases) from both sex and various ages, were collected from various hospitals in Basrah City, Iraq on the year 2001 . Bacteriological investigation for isolation of bacterial pathogens especially Pseudomonas aeruginosa was carried out . Two methods were used to evaluate the anti-bacterial activity of various concentrations of aqueous extracts of leaves of Myrtus communis and Eucalyptus with comparison to 6 antibiotics, these methods were to determine the growth inhibition zones and minimal inhibitory concentration . RESULTS: From a total 92 cases, 78 (84.8%) gave positive cultures . One hundred and fifty eight isolates were identified as bacterial pathogens . Pseudomonas aeruginosa was the predominant bacterial pathogen from 35.9% of the tetracycline covered burns and 42.3% of the non-tetracycline covered burns followed by other bacterial types in various percentages . Aqueous leaves extracts of myrtus communis and eucalyptus gave an excellent effect on bacterial growth and their effects were located within the limits of antibiotic effects . CONCLUSION: Pseudomonas aeruginosa was the predominant bacterial isolate followed by Staphylococcus aureus, then other type of bacterial was isolated in various percentage from each tetracycline covered burns and non-tetracycline covered burns . Most concentrations of the extracts of the studied plants showed a high antibacterial activity against Pseudomonas aeruginosa, and showed significant differences between susceptibility of Pseudomonas aeruginosa isolated from each tetracycline covered burn and non-tetracycline covered burn.

Chin Med J (Engl), 2002 Jul, 115(7), 1099 - 100
Inflammatory reaction and alterations of pulmonary surfactant in Pseudomonas Aeruginosa pneumonia in immunocompromised rats; Qu J et al.; Pulmonary surfactant ( PS ) compromises lipids and surfactant proteins (SP) and lines on the alveolar air-liquid interface . It can reduce surface tension, prevent alveoli from collapse and reduce alveoli edema by disaturated dipalmitoylphosphatidylcholine . It also modulates the pulmonary immunology by SP-A and SP-D . In this study,we established a rat model of immunocompromised host (ICH) with pulmonary infection of Pseudomonas aeruginosa (P . aeruginosa), then studied its pulmonary inflammatory reaction and analyzed the concentration of lipids and SP-A in bronchoalveolar lavage fluid (BALF) during infection.

Eur J Clin Microbiol Infect Dis, 2002 Jul, 21(7), 553 - 6 Epub 2002 Jul 12.
Antiseptic compounds still active against bacterial strains isolated from surgical wound infections despite increasing antibiotic resistance; Giacometti A et al.; The in vitro activities of povidone iodine, potassium peroxymonosulfate, and dimethyldidecylammonium chloride were investigated against 379 nosocomial isolates of Staphylococcus aureus and Pseudomonas aeruginosa responsible for surgical wound infections in patients operated on between July 1995 and June 2001 . Overall, the isolates were inhibited by the antiseptics at concentrations below those used routinely . In spite of increasing resistance to the various antibiotics used to treat surgical wound infections, no significant variation in the susceptibility to antiseptics was demonstrated during this 6-year study.

J Trauma, 2002 Aug, 53(2), 284 - 9; discussion 289-90
Granulocyte colony-stimulating factor: release is not impaired after burn wound infection; Gamelli R et al.; BACKGROUND: The production of granulocyte colony-stimulating factor (G-CSF), the lineage specific essential regulator of neutrophil progenitor cell proliferation and differentiation, has been thought to be impaired in the setting of burn infection . The ability to directly measure murine G-CSF allows the further delineation of the G-CSF response in a clinically relevant model of thermal injury and infection . METHODS: We used a commercially available solid phase enzyme-linked immunoabsorbent assay to quantify G-CSF production after burn wound infection in mice . Bone marrow cells, splenic cells, and serum were obtained from BDF1 mice on day 3 after a 15% total body surface area full-thickness scald burn with or without Pseudomonas aeruginosa burn wound infection . G-CSF production of bone marrow cells or splenic cells and the serum level of G-CSF were measured . A clonogenic assay of bone marrow and spleen granulocyte-macrophage progenitor cells as well as blood leukocyte counts were also performed . RESULTS: After burn sepsis, we noted that G-CSF production of the bone marrow and spleen was significantly increased; the numbers of progenitor cells in bone marrow and spleen were markedly enhanced; serum values of G-CSF were 14 times greater than control values; serum colony-stimulating activity was greater than in control mice; and total blood leukocyte counts were significantly depressed . CONCLUSION: These findings support the notion that granulocytopoietic failure after burn sepsis is not significantly related to defective endogenous G-CSF synthesis . More likely, hyporesponsiveness of granulocyte progenitor cells to G-CSF, changes in the relative balance of granulocyte versus monocyte progenitors within the granulocyte-macrophage progenitor cell compartment, and enhanced release of monocyte lineage specific growth factors are the critical elements responsible for burn infection-induced hematopoietic failure.

EMBO J, 2002 Aug 15, 21(16), 4207 - 18
Structure of the periplasmic domain of Pseudomonas aeruginosa TolA: evidence for an evolutionary relationship with the TonB transporter protein; Witty M et al.; The crystal structure of the C-terminal domain III of Pseudomonas aeruginosa TolA has been determined at 1.9 A resolution . The fold is similar to that of the corresponding domain of Escherichia coli TolA, despite the limited amino acid sequence identity of the two proteins (20%) . A pattern was discerned that conserves the fold of domain III within the wider TolA family and, moreover, reveals a relationship between TolA domain III and the C-terminal domain of the TonB transporter proteins . We propose that the TolA and TonB C-terminal domains have a common evolutionary origin and are related by means of domain swapping, with interesting mechanistic implications . We have also determined the overall shape of the didomain, domains II + III, of P.aeruginosa TolA by solution X-ray scattering . The molecule is monomeric-its elongated, stalk shape can accommodate the crystal structure of domain III at one end, and an elongated helical bundle within the portion corresponding to domain II . Based on these data, a model for the periplasmic domains of P.aeruginosa TolA is presented that may explain the inferred allosteric properties of members of the TolA family . The mechanisms of TolA-mediated entry of bateriophages in P.aeruginosa and E.coli are likely to be similar.

J Bacteriol, 2002 Sep, 184(17), 4912 - 9
LasR, a transcriptional activator of Pseudomonas aeruginosa virulence genes, functions as a multimer; Kiratisin P et al.; The Pseudomonas aeruginosa LasR protein functions in concert with N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C(12)-HSL) to coordinate the expression of target genes, including many genes that encode virulence factors, with cell density . We used a LexA-based protein interaction assay to demonstrate that LasR forms multimers only when 3O-C(12)-HSL is present . A series of LasR molecules containing internal deletions or substitutions in single, conserved amino acid residues indicated that the N-terminal portion of LasR is required for multimerization . Studies performed with these mutant versions of LasR demonstrated that the ability of LasR to multimerize correlates with its ability to function as a transcriptional activator of lasI, a gene known to be tightly regulated by the LasR-3O-C(12)-HSL regulatory system . A LasR molecule that carries a C-terminal deletion can function as a dominant-negative mutant in P . aeruginosa, as shown by its ability to decrease expression of lasB, another LasR-3O-C(12)-HSL target gene . Taken together, our data strongly support the hypothesis that LasR functions as a multimer in vivo.

J Bacteriol, 2002 Sep, 184(17), 4792 - 9
Pseudomonas aeruginosa synthesizes phosphatidylcholine by use of the phosphatidylcholine synthase pathway; Wilderman PJ et al.; Phosphatidylcholine (PC) is a ubiquitous membrane lipid in eukaryotes but has been found in only a limited number of prokaryotes . Both eukaryotes and prokaryotes synthesize PC by methylating phosphatidylethanolamine (PE) by use of a phospholipid methyltransferase (Pmt) . Eukaryotes can synthesize PC by the activation of choline to form choline phosphate and then CDP-choline . The CDP-choline then condenses with diacylglycerol (DAG) to form PC . In contrast, prokaryotes condense choline directly with CDP-DAG by use of the enzyme PC synthase (Pcs) . PmtA was the first enzyme identified in prokaryotes that catalyzes the synthesis of PC, and Pcs in Sinorhizobium meliloti was characterized . The completed release of the Pseudomonas aeruginosa PAO1 genomic sequence contains on open reading frame predicted to encode a protein that is highly homologous (35% identity, 54% similarity) to PmtA from Rhodobacter sphaeroides . Moreover, the P . aeruginosa PAO1 genome encodes a protein with significant homology (39% amino acid identity) to Pcs of S . meliloti . Both the pcs and pmtA homologues were cloned from PAO1, and homologous sequences were found in almost all of the P . aeruginosa strains examined . Although the pathway for synthesizing PC by use of Pcs is functional in P . aeruginosa, it does not appear that this organism uses the PmtA pathway for PC synthesis . We demonstrate that the PC synthesized by P . aeruginosa PAO1 localized to both the inner and outer membranes, where it is readily accessible to its periplasmic, PC-specific phospholipase D.

Eur Respir J Suppl, 2002 Jul, 36, 28s - 39s
Pyogenic bacterial lower respiratory tract infection in human immunodeficiency virus-infected patients; Mayaud C et al.; In human immunodeficiency virus (HIV)-infected patients, bacterial lower respiratory tract infections are the most frequent respiratory diseases . They are frequently the first clinical manifestation of HIV infection . The incidence and severity of bacterial lower respiratory tract infections increase with the degree of immunosuppression . At the acquired immune deficiency syndrome (AIDS) stage, the responsible bacteria and clinical presentation may be atypical . Bacterial pneumonia may be fatal, particularly in AIDS patients, and its occurrence is predictive of a reduced survival time . Pneumococcal vaccine is recommended in patients with a CD4 T-lymphocyte count of > 200 cells mm(-3) and cotrimoxazole (trimethoprim/sulfamethoxazole) in patients with a CD4 T-lymphocyte count of < 200 cells x mm(-3) . Unfortunately, such prophylaxis remains insufficiently prescribed and its protective effect is limited . Highly active antiretroviral treatment has dramatically reduced the incidence of lower respiratory tract infection due to Pseudomonas aeruginosa and opportunistic bacteria . In contrast, successful highly active antiretroviral therapy slightly decreased the risk of bacterial pneumonia due to usual bacteria, even in patients on successful highly active antiretroviral therapy.

DNA Cell Biol, 2002 May-Jun, 21(5-6), 391 - 6
Pseudomonas aeruginosa proteases and corneal virulence; Hobden JA; Pseudomonas aeruginosa is a common cause of corneal infections, particularly among users of soft contact lenses . Previous studies with chemically induced mutants deficient in alkaline protease (AP) or elastase (LasB) suggested that these proteases contributed to the rapid liquifactive stromal necrosis characteristic of P . aeruginosa corneal infections . Because these mutants might harbor other chromosomal changes that could affect virulence, the role of these proteases in the pathogenesis of corneal disease (as well as a second elastase, LasA protease) was reexamined by constructing isogenic mutants deficient only in these enzymes . Allelic exchange was used to construct mutants of P . aeruginosa PAO1-V deficient in AP (PAO1-V AP{ - }), LasB and LasA protease (PDO801 LasB{ - }), or all three proteases (PDO801 TM) . These mutants were then evaluated for virulence using mouse scratch and rabbit intrastromal injection models of corneal disease . Loss of AP significantly increased disease scores in the rabbit (P < 0.030) but not the mouse (P > 0.060) model of infection . Loss of both elastases had no effect on ocular virulence in either animal model of corneal disease (P > 0.100) . The loss of all three proteases significantly decreased disease scores in the rabbit (P < 0.035), but not in the mouse (P > 0.110) . Taken together, these data suggest that AP, LasB, and LasA protease are not essential for initiating or maintaining a corneal infection . Furthermore, AP appears to be an important mediator of pathology depending on the location of the organism within the cornea and whether or not concomitant elastolytic activity is present.

DNA Cell Biol, 2002 May-Jun, 21(5-6), 383 - 90
Pathogenic mechanisms of P . aeruginosa keratitis: a review of the role of T cells, Langerhans cells, PMN, and cytokines; Hazlett LD; The aim of this article is to review our current understanding of the role of cytokines, chemokines, T cells, Langerhans cells, and neutrophils (PMN) and their interactions in vivo in the host response to Pseudomonas aeruginosa ocular challenge . The cellular/cytokine network in vivo has begun to be unraveled, and the data discussed provide substantive evidence for a regulatory role of CD4(+) T cells (Th1 type) contributing directly to persistence of PMN in the cornea of susceptible C57BL/6 (cornea perforates) versus resistant BALB/c (cornea heals) mice . Additionally, in the susceptible mouse model, CD4(+) T cells interact with Langerhans cells and B7/CD28 ligation appears critical for antigen presentation and the susceptibility response . Various cytokines and chemokines (e.g., MIP-1alpha, IL-1beta, MIP-2, IL-12, and IFN-gamma) and their pattern of sustained upregulation after infection in susceptible versus resistant mice also will be discussed in light of an in vivo cytokine network . T-cell-mediated pathogenic mechanisms are of importance in development of the susceptible response to P . aeruginosa ocular infection . In the absence of T-cell infiltration into the cornea, PMN do not persist in the stroma, and cytokines and chemokines are better balanced, resulting in decreased stromal destruction and the resistance response.

Eur Respir J, 2002 Jul, 20(1), 122 - 6
Bronchial reactions to the inhalation of high-dose tobramycin in cystic fibrosis; Nikolaizik WH et al.; It has been established that inhaled tobramycin has a positive effect on respiratory function in Pseudomonas-aeruginosa positive patients with cystic fibrosis (CF) . In a previous study the authors reported that low-dose tobramycin preparations containing the preservative phenol caused significant bronchial obstruction . Recently, high-dose tobramycin preparations with and without preservatives/phenol have become available . To assess the airway response to these preparations flow/volume curves in 12 patients with CF (four males, eight females, mean age+/-SD=19.0+/-7.4 yrs) were measured . The tobramycin preparations: Nebicina 2.0 mL (150 mg, containing the preservative phenol), Distobram 3.0 mL (150 mg, containing preservatives), Tobi 5.0 mL (300 mg), Tobi 2.5 mL (150 mg), and Tobi 5.0 mL, were used after bronchodilator application . Immediately and/or 5 min after the tobramycin inhalations there was a significant fall in lung function with the different preparations . There was no significant difference between preparations with and without preservatives/phenol . The bronchial obstruction was comparable to that observed after the inhalation of low-dose tobramycin and after saline . After 10 min of inhalation, the lung function returned to baseline values . Most patients preferred the Tobi 2.5 mL and disliked the Nebicina preparation due to the unpleasant taste . Preceding treatment with bronchodilators prevented the decline in lung function . Assessment of bronchial response at the first nebulisation of high-dose tobramycin and, in case of significant obstruction, beta-agonists in combination with the antibiotic inhalation are recommended.

Ryumachi, 2002 Jun, 42(3), 610 - 7
{A case report of relapsing polychondritis with an auricular ulcer complicated by systemic sclerosis}; Sugisaki K et al.; A 35-year-old male patient presented with left auricular ulcer following swelling and redness of the left auricle in the beginning of June 2001 . Since 1996, he has been suffering from scleroderma at the fingers and face with Raynaud's phenomenon under a diagnosis of systemic sclerosis . On 1997, partial perforation of his nasal septum was pointed out by an otolaryngologist, which was, however, untreated . He sometimes suffered from swelling and redness in both auricles and from polyarthralgia since 1999 . On June 11th, 2001, hemorrhage and pus discharge under an infection by Pseudomonas aeruginosa started on the painful ulcer in the left auricle, which were not improved by the treatment with antibiotics but improved by an administration of prednisolone at dose of 15 mg/day . Head MRI taken on October 2nd, 2001, showed a partial defect in the nasal septum with a length of 10 mm . The patient was considered as a rare case of a complication of relapsing polychondritis and systemic sclerosis.

Roum Arch Microbiol Immunol, 2001 Jul-Sep, 60(3), 267 - 77
Structure and antimicrobial activity of some new norfloxacin and ofloxacin divalent metal ion complexes; Uivarosi V et al.; The paper presents the studies of some new complexes of norfloxacin (Nf) and ofloxacin (Of) with Cd(II) and Hg(II) . The synthesis, purification and the elemental chemical analysis of the Nf and Of compounds have been performed in order to obtain the chemical formulas . These formulas are confirmed by molecular mass determinations . IR, UV-VIS reflectance spectra were recorded, as well as electric conductometric measurements . These techniques confirm that the obtained compounds are electrolytes and give some hints about their molecular structures . The antimicrobial activity was tested using plates containing Muller-Hinton cultures as well as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa . All compounds and their Nf and Of ligands exert an obvious antimicrobial activity.

Med Sci Monit, 2002 Aug, 8(8), BR289 - 92
Use of a non-contact radiant heat bandage on ischemic dermal infections in an ovine model; d'Audiffret A et al.; BACKGROUND: Chronic non-healing foot wounds are common complication in the diabetic population . Local radiant heat bandage has recently been proposed as an effective adjuvant . The purpose of this study was to evaluate the efficacy of such bandage in controlling infection in an ovine ischemic wound model . MATERIAL/METHODS: Bilateral flank ischemic wounds were created in a total of 42 sheep . 14 sheep were challenged with Pseudomonas aeruginosa (PA), 13 with Escherichia Coli (EC), and 15 with Methicillin resistant staphylococcus aureus (MRSA) . The left flank was designated the treatment side and the right the control side . The radiant heat bandage was applied for a total of 10 days . The animal were then euthanized and the wounds harvested for bacterial quantification . RESULTS: 39 sheep completed the study . Mean bacterial counts were has follows: for MRSA, control 7.6 x 10(5) CFU/gm vs . heated 2.0 x 10(5) CFU/gm (p=0.16); for EC, control 1.1 x 10(6) CFU/gm vs . heated 2.7 x 10(5) CFU/gm (p=0.006); PA, control 1.7 x 10(6) CFU/gm vs . heated 3.9 x 10(9) CFU/gm (p=0.001) . CONCLUSIONS: Non-contact radiant bandages controls bacterial growth in ischemic wounds infected with MRSA or EC and may potentially improve wound healing . Wounds infected with PA should no be submitted to such treatment.

Front Biosci, 2002 Oct 01, 7, e354 - 61
Molecular epidemiology of Pseudomonas aeruginosa; Speert DP; Pseudomonas aeruginosa is a serious opportunistic pathogen in certain compromised hosts, such as those with cystic fibrosis, thermal burns and cancer . It also causes less severe noninvasive disease, such as otitis externa and hot tub folliculitis, in normal hosts . P . aeruginosa is phenotypically very unstable, particularly in patients with chronic infection . Phenotypic typing techniques are useful for understanding the epidemiology of acute infections, but they are limited by their discriminatory power and by their inability to group isolates that are phenotypically unrelated but genetically homologous . Molecular typing techniques, developed over the past decade, are highly discriminatory and are useful for typing strains from patients with chronic infection where the bacterial phenotype is unstable; this is particularly true in cystic fibrosis, where patients often are infected with the same strain for several decades, but the bacteria undergo phenotypic alteration . Molecular typing techniques, which have proven useful in typing P . aeruginosa for epidemiological purposes, include pulsed field gel electrophoresis, restriction fragment length polymorphic DNA analysis, random amplified polymorphic DNA analysis, repetitive extrapalindromic PCR analysis, and multilocus sequence typing . These methods are generally only available in specialized laboratories, but they should be used when data from phenotypic typing analysis are ambiguous or when phenotypic methods are unreliable, such as in cystic fibrosis.

Phytother Res, 2002 May, 16(3), 292 - 4
Coleonema album: studies of the pharmacological action on smooth muscle in vitro and antimicrobial action of its essential oil; Lis-Balchin M et al.; The mode of action of the essential oil of Coleonema album, a South African species from the Cape region, was studied on smooth muscle in vitro using field-stimulated guinea-pig ileum . The oil produced an initial spasmogenic activity followed by spasmolysis, both actions being dose-dependent . The spasmolytic action was post-synaptic and not atropine-like and was unaffected by the adrenoceptor blockers phentolamine and propranolol . There was no evidence for the involvement of either cyclic guanosine monophosphate or cyclic adenosine monophosphate in the spasmolytic activity . The oil had some calcium channel blocking activity but this is not the primary explanation for its spasmolytic response . Antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Staphyloccocus aureus and Saccharomyces cerevisiae was very low and totally absent against Enterococcus hirae, suggesting that the oil has no worthwhile potential in this area . However, as the oil has a very disagreeable odour in its undiluted form, it could be a powerful insect repellent, as its local usage suggests, and this remains to be investigated.

Rev Inst Med Trop Sao Paulo, 2002 May-Jun, 44(3), 167 - 9
Pseudomonas aeruginosa septic shock associated with ecthyma gangrenosum in an infant with agammaglobulinemia; Almeida JF et al.; Ecthyma gangrenosum (EG) due to Pseudomonas aeruginosa is a rare and invasive infection that can be associated with agammaglobulinemia . The cornerstone of the treatment is based on prompt recognition with appropriate antibiotic coverage and intravenous immunoglobulin . The authors report a case of EG emphasizing the clinical and therapeutic aspects of this condition.

Crit Care Med, 2002 Aug, 30(8), 1899 - 901
Skin necrosis after extravasation of low-dose vasopressin administered for septic shock; Kahn JM et al.; OBJECTIVE: To describe a case of severe skin necrosis resulting from peripheral intravenous administration of low-dose vasopressin in a patient with catecholamine-resistant septic shock . DESIGN: Case report . SETTING: Medical intensive care unit at the University of Chicago, Chicago, IL . PATIENT: A 46-yr-old female with ventilator-dependent, proliferative-phase acute respiratory distress syndrome complicated by Pseudomonas aeruginosa bacteremia and sepsis . MEASUREMENTS AND MAIN RESULTS: A patient recovering from acute respiratory distress syndrome developed septic shock from Pseudomonas aeruginosa bacteremia while in the medical intensive care unit . Vasopressin (0.04 units/min) was administered through a peripheral venous catheter for hypotension unresponsive to exogenous catecholamines . The patient subsequently developed severe ischemic necrosis of the skin and soft tissue surrounding the catheter site . The vasopressin was stopped, and the skin lesion progressed to bullae formation with extensive superficial erosion . CONCLUSIONS: Peripheral administration of low-dose vasopressin for septic shock should be discouraged because of the risk of ischemic skin complications.

Am J Pathol, 2002 Aug, 161(2), 551 - 64
Pseudomyxoma peritonei is a disease of MUC2-expressing goblet cells; O'Connell JT et al.; Pseudomyxoma peritonei, a syndrome first described by Karl F . Rokitansky in 1842, is an enigmatic, often fatal intra-abdominal disease characterized by dissecting gelatinous ascites and multifocal peritoneal epithelial implants secreting copious globules of extracellular mucin . Although past interest in the syndrome has focused on the questions of the site of origin (appendix versus ovary), mechanisms of peritoneal spread (multicentricity, redistribution phenomenon, or metastasis), and the degree of malignant transformation present (adenoma, borderline tumor, or carcinoma), another important question is the mechanism behind the accumulation of extracellular mucin, the real cause of the disease's morbidity and mortality irrespective of the site of origin, mechanism of peritoneal spread, or transformed status of its epithelium . Taking advantage of the recently cloned human mucin genes, we decided to investigate this question . Our studies revealed that pseudomyxoma peritonei is a disease of MUC2-expressing goblet cells . These cells also express MUC5AC but the latter mucin is not specific for pseudomyxoma peritonei . MUC2 expression accounts for the voluminous deposits of extracellular mucin (mucin:cell ratios exceeding 10:1) and distinguishes pseudomyxoma peritonei secondarily involving the ovary from primary ovarian mucinous tumors with peritoneal implants . Because mucinous tumors of the appendix similarly express MUC2, the MUC2 expression profile also supports an appendiceal rather than ovarian origin for pseudomyxoma peritonei . Increased steady-state mRNA is observed in pooled cases of pseudomyxoma peritonei but does not occur on the basis of gene rearrangement or gene amplification . Primary epithelial cell cultures obtained from pseudomyxoma peritonei express MUC2 whose levels can be epigenetically regulated . These lines up-regulate MUC2 expression in response to both methylation inhibition by 5-azacytidine and exposure to Pseudomonas aeruginosa lipopolysaccharide, both of whose effects can be suppressed by genistein pretreatment . Both immunocytochemical as well as in situ hybridization studies with ancillary digital image analysis reveal that MUC2 expression in cases of pseudomyxoma peritonei is independent of the degrees of malignant transformation that are present and, in fact, reflects the constitutive levels of expression observed in normal goblet cells of the appendix . Extracellular mucin accumulates dramatically in pseudomyxoma peritonei because the number of MUC2-secreting cells dramatically increase and because this MUC2 has no place to drain . These studies suggest that pseudomyxoma peritonei should be regarded as a disease of MUC2-expressing goblet cells whose MUC2 expression might be susceptible to pharmacological targeting.

Biochem J, 2002 Nov 1, 367(Pt 3), 601 - 8
A re-evaluation of the role of histidine-426 within Pseudomonas aeruginosa exotoxin A; Roberts TM et al.; CRM66 (cross-reactive 66 kDa protein) is an inactive mutant form of Pseudomonas aeruginosa exotoxin A that has been isolated from a mutant strain of P . aeruginosa derived from nitrosoguanidine-based mutagenesis . The mutation within this enzyme toxin was previously identified as H426Y and it was shown to possess significantly reduced enzymic activity . Furthermore, it was previously suggested that His-426 may directly participate in the catalytic mechanism of the exotoxin A enzyme and that it may also play an important role in the binding of the protein substrate of exotoxin A, a critical protein factor in eukaryotic protein translation known as elongation factor-2 . In order to more thoroughly characterize the role of His-426 in the enzyme mechanism of exotoxin A, amino acid substitutions were made within helix 1 of the enzyme domain in the vicinity of the His-426 residue . Analysis of the site-directed mutagenesis results involving kinetic and protein structural integrity measurements revealed that His-426 H-bonds to Tyr-502 and that replacement of His-426 with polar substitutions leads to structural alterations of the enzyme's folded conformation . Furthermore, it was shown that His-426 is not important for the binding of either of the two substrates of exotoxin A, NAD(+) or elongation factor-2 . In summary, these data show that His-426 is not an active-site residue and that it is not important for substrate binding or orientation, but that it plays an important structural role in helping to maintain the folded conformation of the enzyme toxin . Therefore, the role of His-426 would seem to be to tether helix 1 to the main body of the enzyme, and mutations resulting in the disruption of this region of the enzyme result in a significantly impaired enzyme.

Am J Respir Crit Care Med, 2002 Aug 1, 166(3), 356 - 61
Detecting Stenotrophomonas maltophilia does not reduce survival of patients with cystic fibrosis; Goss CH et al.; Stenotrophomonas maltophilia is a gram-negative bacterium that has been cultured with increasing prevalence from the sputum of patients with cystic fibrosis (CF) . We conducted a cohort study, using the Cystic Fibrosis Foundation Registry, to assess the effect of S . maltophilia on survival . We studied all patients in this registry from 1991 to 1997 who were older than 6 years of age, were S . maltophilia negative in the first year of enrollment, and had their CF diagnosed before the age of 45 years (n = 19,255 in the study) . We compared patients who acquired S . maltophilia with those who did not, using survival analysis . A total of 1,673 (8.7%) had at least one sputum sample positive for S . maltophilia . Compared with patients without S . maltophilia, those patients positive for S . maltophilia had the following baseline characteristics before detection: lower FEV(1) % pred (p < 0.001); older (p = 0.001); more likely to be female (p = 0.003); and more pulmonary exacerbations (p < 0.001), outpatient visits (p < 0.002), and total hospitalizations (p < 0.001) . After controlling for differences in severity of disease and coinfection with Pseudomonas aeruginosa, the hazard ratio associated with S . maltophilia detection was 0.89 (95% confidence interval, 0.75-1.05) . Although patients with CF who acquire S . maltophilia have more advanced disease than those who do not acquire this organism, detection does not independently affect short-term survival (3 years).

J Biochem (Tokyo), 2002 Aug, 132(2), 353 - 8
Production, properties and specificity of a new bacterial L-fucose- and D-arabinose-binding lectin of the plant aggressive pathogen Ralstonia solanacearum, and its comparison to related plant and microbial lectins; Sudakevitz D et al.; The worldwide distributed plant aggressive pathogen Ralstonia solanacearum, which causes lethal wilt in many agricultural crops, produces a potent L-fucose-binding lectin (RSL) exhibiting sugar specificity similar to that of PA-IIL of the human aggressive opportunistic pathogen Pseudomonas aeruginosa . Both lectins show L-fucose > L-galactose > D-arabinose > D-mannose specificity, but the affinities of RSL to these sugars are substantially lower . Unlike Ulex europaeus anti-H lectin, but like PA-IIL and Aleuria aurantia lectin (AAL), RSL agglutinates H-positive human erythrocytes regardless of their type, O, A, B, or AB, and animal erythrocytes (papain-treated ones more strongly than untreated ones) . It also interacts with H and Lewis chains in the saliva of "secretors" and "nonsecretors." RSL purification is easier than that of PA-IIL since R . solanacearum extracts do not contain a galactophilic PA-IL-like activity . Mass spectrometry and 35 N-terminal amino acid sequencing enabled identification of the RSL protein (subunit approximately 9.9 kDa, approximately 90 amino acids) in the complete genome sequence of this bacterium . Despite the greater phylogenetic proximity of R . solanacearum to P . aeruginosa, and the presence of a PA-IIL-like gene in its genome, the RSL structure is not related to that of PA-IIL, but to that of the fucose-binding lectin of the mushroom (fungus) Aleuria aurantia, which like the two bacteria is a soil inhabitant.

Microbiol Immunol, 2002, 46(6), 391 - 5
High-level fluoroquinolone resistance in Pseudomonas aeruginosa due to interplay of the MexAB-OprM efflux pump and the DNA gyrase mutation; Nakajima A et al.; Fluoroquinolone resistance in Pseudomonas aeruginosa is mainly attributable to the constitutive expression of the xenobiotic efflux pump and mutation in DNA gyrase or topoisomerase IV . We constructed cells with a double-mutation in gyrA and mexR encoding DNA gyrase and repressor for the mexAB-oprM operon, respectively . The mutant showed 1,024 times higher fluoroquinolone resistance than cells lacking the MexAB-OprM . Cells with a single mutation in gyrA and producing a wild-type level of the MexAB-OprM efflux pump showed 128 times higher fluoroquinolone resistance than cells lacking the MexAB-OprM . In contrast, a single mutation in gyrA or mexR caused only 4 and 64 times higher resistance, respectively . These findings manifested the interplay between the MexAB-OprM efflux pump and the target mutation in fluoroquinolone resistance.

Plasmid, 2002 May, 47(3), 216 - 23
Host-specific incompatibility by 9-bp direct repeats indicates a role in the maintenance of broad-host-range plasmid RK2; Wilson JW et al.; Broad-host-range incompatibility group P (IncP) plasmids RK2 and R751 have 9-bp direct repeats (DR) of unknown function located between their kilC and kilE loci . The nucleotide sequences of the 9-bp repeats are different for RK2 (an IncPalpha group plasmid) and R751 (IncPbeta group), but both DR regions are organized similarly, including an 11-bp spacer with identical 5'-CGCCA-3' cores and an adjacent binding site for KorB, a known partition protein and transcriptional repressor . The occurrence of similarly arranged DR elements with different repeat sequences is suggestive of an important plasmid-specific function for the DR regions . Here we show that the cloned RK2 DR region in trans to RK2 exhibits a host-specific incompatibility phenotype, in which RK2 is destabilized in Pseudomonas aeruginosa but not in Escherichia coli . Incompatibility was not dependent on the adjacent KorB-binding site . Deletion of the kilE locus, which is required for stable maintenance in P . aeruginosa, did not abolish DR-mediated incompatibility . Precise deletion of DR from RK2 had no effect on maintenance but eliminated sensitivity to DR in trans, showing that incompatibility requires DR to be present on both plasmids . These results raise the possibility that the DR region may be involved in a plasmid maintenance system for P . aeruginosa that is independent of the known stability functions on RK2.

Biochim Biophys Acta, 2002 Aug 19, 1577(1), 155 - 8
Functional expression of murine LRP1 requires correction of Lrp1 cDNA sequences; Smeijers L et al.; Here, we describe the reconstruction of a functional 14 kbp full-length murine Lrp1 cDNA from overlapping partial cDNAs, which were described before {Biochim . Biophys . Acta 1173 (1993) 71} . The reconstructed full-length cDNA needed sequence correction (by mutagenesis) due to nucleotide errors present in the underlying partial cDNAs . These mistakes compromised the proteolytical maturation of the LRP precursor (4545 aa) into its alpha- and beta-subunits . To identify these mistakes initially, detailed sequence analyses and comparison of genomic and cDNA sequences of different murine strains proved to be necessary to obtain correct wild-type sequences . Comparison of Lrp1 cDNA sequences of CBA mice with Lrp1 genomic exon sequences of 129P3/J mice (like in man 89 exons) revealed only 24 nucleotide differences within about 14.8 kbp . Only 1 out of 23 nucleotide differences in the protein coding region affected an amino acid residue: Thr versus Ala at amino acid residue position 2642 in 129P3/J and CBA, respectively . After correction by mutagenesis, both a 129P3/J and a CBA-based version of a full-length wild-type Lrp1 cDNA were functionally expressed in an LRP-deficient mutant CHO cell line . Transient expression showed the expected maturation of the LRP precursor into its two subunits . Furthermore, stable transfection restored the sensitivity to exposure to Pseudomonas aeruginosa toxin A (PEA) . Since LRP is the unique receptor for this toxin, this indicates that the toxin could enter the cells after binding to and endocytosis by its genuine receptor . This murine LRP expression system will be instrumental in future experimental dissection of this multifunctional receptor.

J Clin Microbiol, 2002 Aug, 40(8), 2772 - 8
Genetic analysis of Pseudomonas aeruginosa isolates from the sputa of Australian adult cystic fibrosis patients; Anthony M et al.; Genetic investigations were carried out with 50 phenotypically selected strains of Pseudomonas aeruginosa from 18 patients attending an Australian cystic fibrosis (CF) center . The isolates were analyzed by restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE) . Phylogenetic analysis of the macrorestriction patterns showed rates of genetic similarity ranging from 76 to 100%; 24 (48%) of the strains from 11 patients had greater than 90% similarity . A dominant strain emerged: 15 isolates from seven patients had identical PFGE patterns, and 4 other isolates were very closely related . The 50 isolates were grouped into 21 pulsotypes on the basis of visual delineation of a three-band difference . Ten of the 18 (56%) patients were infected with clonal or subclonal strains . Sequence analysis of PCR products derived from the mucA gene showed 20 mutations, with the number of mutations in individual isolates ranging from 1 to 4; 19 of these changes are reported here for the first time . Potentially functional changes were found in 22 (44%) isolates . Eight changes (five transversions and three single base deletions) led to premature stop codons, providing support for the presence of mucA mutations as one pathway to mucoidy . There was a trend toward an association between the dominant strain and lack of potentially functional mucA mutations (P = 0.09 by the chi(2) test) but no relationship between genotype and phenotype . This is the first study of genetic variation in P . aeruginosa isolates from adult Australian CF patients . The findings highlight the need for further investigations on the transmissibility of P . aeruginosa in CF patients.

Acta Paediatr Taiwan, 2002 May-Jun, 43(3), 157 - 61
Community-acquired Pseudomonas aeruginosa pneumonia complicated with loculated empyema in an infant with selective IgA deficiency; Chen SM et al.; Pseudomonas aeruginosa is widely prevalent in the hospital environment, especially in intensive care units . Selective IgA deficiency is characterized by a serum IgA level less than 5 mg/dl with no deficiency of other immunoglobulins . The occurrence of community-acquired P . aeruginosa pneumonia with empyema is rare in pediatric patients . We present a 10-month-old male infant who was referred due to persistent fever and progressive respiratory distress for 1 week . A chest radiograph revealed a right lobar pneumonia with pleural effusion . P . aeruginosa that was subsequently isolated from both blood and pleural effusion cultures . The patient received treatment with ceftazidime and intrapleural instillation of urokinase to promote drainage of empyema . Subsequent immunological screening revealed a very low serum IgA level (<5 mg/dl) . We present our experience in successfully treating a loculated empyema with intrapleural instillation of urokinase in an infant . It is also important for pediatricians to be aware that they should be alert for the patient who present with respiratory infections due to unusual organisms . An advanced immunological study to investigate the underlying disorders in these patients is mandatory.

Lab Anim, 2002 Jul, 36(3), 291 - 312
Murine models of chronic Pseudomonas aeruginosa lung infection; van Heeckeren AM et al.; The animal model of chronic bronchopulmonary infection using agarose beads laden with Pseudomonas aeruginosa is frequently utilized in cystic fibrosis research, though it is challenging to perform it in mice . This paper reports the most successful methods for the creation of this model . Transtracheal insertion of a 22 G 1" over-the-needle intravenous catheter to preferentially inoculate the right mainstem bronchus using tribromoethanol anaesthesia administered i.p . was better for a successful surgical outcome compared, respectively, to the use of a 27 G (1/2)" needle, bilateral inoculation or an anaesthetic cocktail of xylazine, acepromazine and ketamine administered i.p . Bilateral infection was associated with higher mortality, greater weight loss and higher levels of bronchoalveolar cytokine concentration, compared to mice infected primarily in the right lung . Mucoid clinical strain PA M57-15 was preferred since mucoid clinical strain PA 2192 led to comparatively more severe lesions and higher mortality . Using the same operator for a given task reduced the variability inherent in this model, illustrated using outcome measures such as gross lung pathology . The response of mice inoculated with P . aeruginosa-laden agarose beads was characterized by bronchopulmonary inflammation, high production of cytokines, and significant weight loss; whereas the response to infection with free-living bacteria was characterized by pneumonia, lower production of cytokines and weight loss . The use of free P . aeruginosa pre-mixed with sterile agarose beads may be considered as an alternative to the use of P . aeruginosa-laden agarose beads, since the histopathological features were similar, though further characterization is needed to evaluate its utility as an adequate model of cystic fibrosis.

Science, 2002 Jul 26, 297(5581), 623 - 6
A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity; Kim DH et al.; A genetic screen for Caenorhabditis elegans mutants with enhanced susceptibility to killing by Pseudomonas aeruginosa led to the identification of two genes required for pathogen resistance: sek-1, which encodes a mitogen-activated protein (MAP) kinase kinase, and nsy-1, which encodes a MAP kinase kinase kinase . RNA interference assays and biochemical analysis established that a p38 ortholog, pmk-1, functions as the downstream MAP kinase required for pathogen defense . These data suggest that this MAP kinase signaling cassette represents an ancient feature of innate immune responses in evolutionarily diverse species.

Annu Rev Microbiol, 2002, 56, 17 - 38 Epub 2002 Jan 30.
Function of pseudomonas porins in uptake and efflux; Hancock RE et al.; Porins are proteins that form water-filled channels across the outer membranes of Gram-negative bacteria and thus make this membrane semipermeable . There are four types of porins: general/nonspecific porins, substrate-specific porins, gated porins, and efflux porins (also called channel-tunnels) . The recent publication of the genomic sequence of Pseudomonas aeruginosa PAO1 has dramatically increased our understanding of the porins of this organism . In particular this organism has 3 large families of porins: the OprD family of specific porins (19 members), the OprM family of efflux porins (18 members), and the TonB-interacting family of gated porins (35 members) . These familial relationships underlie functional similarities such that well-studied members of these families become prototypes for other members . We summarize here the latest information on these porins.

J Bacteriol, 2002 Aug, 184(16), 4374 - 83
Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response; Ferrandez A et al.; Pseudomonas aeruginosa, a gamma-proteobacterium, is motile by means of a single polar flagellum and is chemotactic to a variety of organic compounds and phosphate . P . aeruginosa has multiple homologues of Escherichia coli chemotaxis genes that are organized into five gene clusters . Previously, it was demonstrated that genes in cluster I and cluster V are essential for chemotaxis . A third cluster (cluster II) contains a complete set of che genes, as well as two genes, mcpA and mcpB, encoding methyl-accepting chemotaxis proteins . Mutations were constructed in several of the cluster II che genes and in the mcp genes to examine their possible contributions to P . aeruginosa chemotaxis . A cheB2 mutant was partially impaired in chemotaxis in soft-agar swarm plate assays . Providing cheB2 in trans complemented this defect . Further, overexpression of CheB2 restored chemotaxis to a completely nonchemotactic, cluster I, cheB-deficient strain to near wild-type levels . An mcpA mutant was defective in chemotaxis in media that were low in magnesium . The defect could be relieved by the addition of magnesium to the swarm plate medium . An mcpB mutant was defective in chemotaxis when assayed in dilute rich soft-agar swarm medium or in minimal-medium swarm plates containing any 1 of 60 chemoattractants . The mutant phenotype could be complemented by the addition of mcpB in trans . Overexpression of either McpA or McpB in P . aeruginosa or Escherichia coli resulted in impairment of chemotaxis, and these cells had smooth-swimming phenotypes when observed under the microscope . Expression of P . aeruginosa cheA2, cheB2, or cheW2 in E . coli K-12 completely disrupted wild-type chemotaxis, while expression of cheY2 had no effect . These results indicate that che cluster II genes are expressed in P . aeruginosa and are required for an optimal chemotactic response.

Mol Microbiol, 2002 Aug, 45(3), 755 - 68
Pseudomonas aeruginosa RoxR, a response regulator related to Rhodobacter sphaeroides PrrA, activates expression of the cyanide-insensitive terminal oxidase; Comolli JC et al.; The facultative anaerobe Pseudomonas aeruginosa has multiple aerobic electron transport pathways, one of which is terminated by a cyanide-insensitive oxidase (CIO) . This study characterizes a P . aeruginosa two-component system that regulates CIO production . The response regulator of this system (RoxR) has significant amino acid sequence similarity to PrrA of Rhodobacter sphaeroides and related proteins in other alpha-proteobacteria . In heterologous complementation analysis, R . sphaeroides PrrA rescued the growth defect of a P . aeruginosa mutant lacking RoxR, and RoxR enabled photosynthetic growth of an R . sphaeroides PrrA mutant . Also, RoxR could substitute for PrrA in activating transcription in vitro, demonstrating that these proteins are functional homologues . P . aeruginosa strains lacking RoxR or the sensor kinase (RoxS) were more sensitive than wild type to the respiratory inhibitors cyanide and azide . The phenotypes of these mutant strains correlated with reduced cyanide-insensitive O2 utilization and less cyanide-dependent expression of the locus encoding the CIO (cioAB) . The ability of purified RoxR to bind to the cioAB promoter region also suggests that this protein acts directly to regulate cioAB transcription . Therefore, RoxR appears to play a role in regulating the transcription of loci for P . aeruginosa energy-generating enzymes similar to that of its homologues in alpha-proteobacteria.

Microb Pathog, 2002 Jun, 32(6), 287 - 95
Role and activation of type III secretion system genes in Pseudomonas aeruginosa-induced Drosophila killing; Fauvarque MO et al.; Pseudomonas aeruginosa strains PAO1 and CHA showing type III system-dependent cytotoxicity towards macrophages ex vivo are able to induce rapid death of adult fly Drosophila melanogaster accompanied by bacterial multiplication to high-titers . The role of P . aeruginosa type III secretion system in rapid fly killing was demonstrated here by using several isogenic CHA mutants, selectively affected in this system . The activation of P . aeruginosa pexsCBA, the regulatory operon of the type III system, and the activation of the Drosophila gene diptericin, showed the host-pathogen recognition during infection process.

Biochemistry, 2002 Jul 30, 41(30), 9680 - 7
ADP-ribosylation and functional effects of Pseudomonas exoenzyme S on cellular RalA; Fraylick JE et al.; Exoenzyme S (ExoS) is a bifunctional virulence factor directly translocated into eukaryotic cells by the type III secretory process of Pseudomonas aeruginosa . Bacterial translocation of ExoS into epithelial cells is associated with diverse effects on cell function, including inhibition of growth, alterations in cell morphology, and effects on adherence processes . Preferred substrates of the ADP-ribosyltransferase (ADPRT) portion of ExoS include low molecular weight G-proteins (LMWG-proteins) in the Ras family . In examining the ADP-ribosylation and functional effects of ExoS on RalA, ExoS was found to ADP-ribosylate endogenous RalA and recombinant RalADeltaCAAX at multiple sites, with Arg52 identified as the preferred site of ADP-ribosylation . The binding of RalA to the Ral binding domain (RBD) of its downstream effector, RalBP1, was inhibited by bacterially translocated ExoS, indicating an effect of ExoS on cellular RalA function . In vitro analyses confirmed that ADP-ribosylation of RalA directly interfered with its ability to bind to the RBD of RalBP1 . The studies support the fact that RalA is a cellular substrate of bacterially translocated ExoS and that ADP-ribosylation by ExoS affects RalA interaction with its downstream effector, RalBP1.

Biochemistry, 2002 Jul 30, 41(30), 9637 - 45
Allosterism and cooperativity in Pseudomonas aeruginosa GDP-mannose dehydrogenase; Naught LE et al.; GDP-Mannose dehydrogenase catalyzes the formation of GDP-mannuronic acid, which is the monomeric unit from which the polysaccharide alginate is formed . Alginate is secreted by the pathogenic bacterium Pseudomonas aeruginosa and is believed to play an important role in the bacteria's resistance to antibiotics and the host immune response . We have characterized the kinetic behavior of GDP-mannose dehydrogenase in detail . The enzyme displays cooperative behavior with respect to NAD(+) binding, and phosphate and GMP act as allosteric effectors . Binding of the allosteric effectors causes the Hill coefficient for NAD(+) binding to decrease from 6 to 1, decreases K(1/2) for NAD(+) by a factor of 10, and decreases V(max) by a factor of 2 . The cooperative binding of NAD(+) is also sensitive to pH; deprotonation of two residues with identical pK's of 8.0 is required for maximally cooperative behavior . The kinetic behavior of GDP-mannose dehydrogenase suggests that it must be at least hexameric under turnover conditions; however, dynamic light-scattering measurements do not provide a clear determination of the size of the active enzyme complex.

Pharm Res, 2002 Jun, 19(6), 818 - 24
Physicochemical characterization of hexetidine-impregnated endotracheal tube poly(vinyl chloride) and resistance to adherence of respiratory bacterial pathogens; Jones DS et al.; PURPOSE: Ventilator-associated pneumonia is a frequent cause of mortality in intensive care patients . This study describes the physicochemical properties of hexetidine-impregnated poly(vinyl chloride) (PVC) endotracheal tube (ET) biomaterials and their resistance to microbial adherence (Staphylococcus aureus and Pseudomonas aeruginosa) . METHODS: PVC emulsion was cured in the presence of hexetidine (0-20% w/w) and was characterized in terms of drug release, surface properties (i.e., microrugosity/contact angle), mechanical (tensile) properties, and resistance to microbial adherence . RESULTS: Under sink conditions, hexetidine release from PVC was diffusion-controlled . Increasing the concentration of hexetidine from 1% to 10% (w/w) (but not from 10% to 20% w/w) increased the subsequent rate of drug release . In general, increasing the concentration of hexetidine decreased both the tensile properties and hydrophobicity, yet increased PVC microrugosity . Following hexetidine release (21 days), the surface properties were similar to those of native PVC . The resistance of hexetidine-containing PVC (1% or 5%) to microbial adherence (following defined periods of drug release) was greater than that of native PVC and was constant over the examined period of hexetidine release . CONCLUSIONS: ET PVC containing 1% (w/w) hexetidine offered an appropriate balance between suitable physicochemical properties and resistance to microbial adherence . This may offer an approach with which to reduce the incidence of ventilator-associated pneumonia . KEY WORDS: PVC endotracheal tube; hexetidine; characteriza-

Biochem J, 2002 Nov 15, 368(Pt 1), 91 - 100
Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles; Rinaldi AC et al.; The temporins are a family of small, linear antibiotic peptides with intriguing biological properties . We investigated the antibacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH2), isolated from the skin of the European red frog Rana temporaria . The peptide displayed the highest activity of temporins studied to date, against both human erythrocytes and bacterial and fungal strains . At variance with other known temporins, which are mainly active against Gram-positive bacteria, temporin L was also active against Gram-negative strains such as Pseudomonas aeruginosa A.T.C.C . 15692 and Escherichia coli D21 at concentrations comparable with those that are microbiocidal to Gram-positive bacteria . In addition, temporin L was cytotoxic to three different human tumour cell lines (Hut-78, K-562 and U-937), causing a necrosis-like cell death, although sensitivity to the peptide varied markedly with the specific cell line tested . A study of the interaction of temporin L with liposomes of different lipid compositions revealed that the peptide causes perturbation of bilayer integrity of both neutral and negatively charged membranes, as revealed by the release of a vesicle-encapsulated fluorescent marker, and that the action of the peptide is modulated to some extent by membrane lipid composition . In particular, the presence of negatively charged lipids in the model bilayer inhibits the lytic power of temporin L . We also show that the release of fluorescent markers caused by temporin L is size-dependent and that the peptide does not have a detergent-like effect on the membrane, suggesting that perturbation of bilayer organization takes place on a local scale, i.e . through the formation of pore-like openings.

Biochem J, 2002 Nov 1, 367(Pt 3), 617 - 28
Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo; Henriksson ML et al.; Intracellular targeting of the Pseudomonas aeruginosa toxins exoenzyme S (ExoS) and exoenzyme T (ExoT) initially results in disruption of the actin microfilament structure of eukaryotic cells . ExoS and ExoT are bifunctional cytotoxins, with N-terminal GTPase-activating protein (GAP) and C-terminal ADP-ribosyltransferase activities . We show that ExoS can modify multiple GTPases of the Ras superfamily in vivo . In contrast, ExoT shows no ADP-ribosylation activity towards any of the GTPases tested in vivo . We further examined ExoS targets in vivo and observed that ExoS modulates the activity of several of these small GTP-binding proteins, such as Ras, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42 . We suggest that ExoS is the major ADP-ribosyltransferase protein modulating small GTPase function encoded by P . aeruginosa . Furthermore, we show that the GAP activity of ExoS abrogates the activation of RhoA, Cdc42 and Rap1.

J Med Microbiol, 2002 Jul, 51(7), 620 - 2
Community-acquired Pseudomonas aeruginosa sacro-iliitis in a previously healthy patient; Calza L et al.; Pyogenic sacro-iliitis is an uncommon osteo-articular infection that occurs usually in immunocompromised patients and is associated with gram-positive cocci . It is very rarely linked with a gram-negative aetiology . The first case of Pseudomonas aeruginosa sacro-iliitis is described, which occurred in a previously healthy young man, without history of prior traumatic events, hospitalisation or chronic underlying disease.

J Med Microbiol, 2002 Jul, 51(7), 615 - 9
A clinical index predicting mortality with Pseudomonas aeruginosa bacteraemia; Aliaga L et al.; The aim of this study was to define risk factors associated with mortality in Pseudomonas aeruginosa bactaeremia and to combine them in a clinical index predicting the risk of death . The study investigated 125 consecutive episodes of P . aeruginosa bacteraemia at this hospital . Crude mortality was 34%, corresponding to 43 patients who died, with 67% of deaths, directly attributable to bacteraemia . A regression logistic model identified five variables that were independently and significantly associated with an increased risk of death: 1) hospitalisation in the intensive care unit; 2) coagulopathy; 3) septic shock; 4) age > or = 65 years; and 5) the clinical condition of the patient . These variables were as recorded at the time that the first positive blood culture was obtained . The sensitivity and specificity of a prediction of death based on the model were 84% and 85%, respectively . An index score, calculated from these variables, divided patients into three groups with increasing likelihood of mortality resulting from P . aeruginosa bacteraemia.

J Surg Res, 2002 Jul, 106(1), 86 - 98
Anti-ETA IgG neutralizes the effects of Pseudomonas aeruginosa exotoxin A; Fogle MR et al.; BACKGROUND: The opportunistic pathogen Pseudomonas aeruginosa causes severe infections in immunocompromised hosts . Among P . aeruginosa-infected burn patients, mortality may reach as high as 50% . Due to their immunocompromised status, burn patients may benefit from passive immunotherapy against infection . As a potential multivalent immunoglobulin therapy, specific polyclonal antibodies against four P . aeruginosa virulence factors, including exotoxin A (ETA), were prepared . MATERIALS AND METHODS AND RESULTS: In this study, we analyzed the ability of ETA antibody (ETA-Ab) to neutralize the in vivo effects of ETA . Adult mice injected with purified ETA suffered 100% mortality . The cytosolic DNA of their hepatocytes was fragmented, indicating ETA induction of apoptosis . In addition, multiprobe RNase protection assays showed that ETA upregulates the expression of the genes for proinflammatory cytokines as well as apoptosis genes in the livers of ETA-injected mice . Treatment with ETA-Ab prior to ETA injection prevented mortality, ETA-induced hepatocyte DNA fragmentation, and upregulation of the cytokine and apoptosis-related genes . The role of ETA during P . aeruginosa infection of the burn wound was examined by determining the in vivo virulence of P . aeruginosa PA103 and its isogenic, ETA-deficient mutant PA103Omega::toxA using the thermally injured mouse model . The lethality, local spread, and systemic spread of PA103Omega::toxA were significantly reduced compared to PA103 . CONCLUSION: These results suggest that (1) ETA induces apoptosis in hepatocytes, (2) specific cytokines are produced in response to ETA, (3) ETA-Ab neutralizes these effects, and (4) ETA contributes to the spread of P . aeruginosa during burn wound infection.

Int J Antimicrob Agents, 2002 Jul, 20(1), 44 - 9
Bacterial strain-independent AUC/MIC and strain-specific dose-response relationships reflecting comparative fluoroquinolone anti-pseudomonal pharmacodynamics in an in vitro dynamic model; Lubenko IY et al.; To compare the antimicrobial effects (AMEs) of two quinolones in terms of the AUC/MIC- and dose (D)-response relationships, five differentially susceptible clinical isolates of Pseudomonas aeruginosa were exposed to decreasing concentrations of ciprofloxacin (two 12-h doses with T(1/2) = 4 h) and trovafloxacin (a single dose with T(1/2) = 9.2 h) . The simulated AUC/MICs of ciprofloxacin ranged from 58 to 932 and those of trovafloxacin, from 54 to 466 h . The intensity of the AME (I(E)) correlated well with log AUC/MIC for both ciprofloxacin and trovafloxacin (r(2) = 0.99 and 0.97, respectively) in a strain-independent fashion . At a given AUC/MIC ratio, AMEs of trovafloxacin were greater than ciprofloxacin . However, based on the respective I(E)-logD curves, 200 mg trovafloxacin produced a slightly greater AME than 2 x 500 mg ciprofloxacin only with the most susceptible P . aeruginosa . With the less susceptible P . aeruginosa ciprofloxacin was more efficient than trovafloxacin . This study suggests that both bacterial strain-independent AUC/MIC- and the respective strain-specific dose-response relationships of the AME are important for comprehensive pharmacodynamic evaluation of antimicrobial agents.

FEMS Microbiol Lett, 2002 Jul 16, 213(1), 73 - 9
Pseudomonas aeruginosa internalization by corneal epithelial cells involves MEK and ERK signal transduction proteins; Evans DJ et al.; Invasion of epithelial cells represents a potential pathogenic mechanism for Pseudomonas aeruginosa . We explored the role of mitogen-activated protein kinase kinases (MEK 1/2) and the extracellular signal-regulated kinases (ERK 1/2) in P . aeruginosa invasion . Treatment of corneal epithelial cells with MEK inhibitors, PD98059 (20 microM) or UO126 (100 microM), reduced P . aeruginosa invasion by approximately 60% without affecting bacterial association with the cells (P=0.0001) . UO124, a negative control for UO126, had no effect on bacterial internalization . Infection of cells with an internalization-defective flhA mutant of P . aeruginosa was associated with less ERK 1/2 tyrosine phosphorylation than infection with wild-type invasive P . aeruginosa . An ERK-2 inhibitor, 5-iodotubercidin (20 microM), reduced P . aeruginosa invasion by approximately 40% (P=0.035) . Together, these data suggest that P . aeruginosa internalization by epithelial cells involves a pathway(s) that includes MEK and ERK signaling proteins.

FEMS Microbiol Lett, 2002 Jul 16, 213(1), 1 - 6
Solubilization of zinc salts by a bacterium isolated from the air environment of a tannery; Fasim F et al.; Airborne bacteria isolated from a tannery air environment were screened for the property of solubilization of insoluble zinc oxide and zinc phosphate . Out of 10 strains tested, a strain of Pseudomonas aeruginosa (CMG 823) showed the best solubilization and solubilized both zinc oxide and zinc phosphate . Colonies of the bacterium produced clear haloes on solid medium which contained these insoluble metal compounds, but only when glucose was provided as a carbon source . Solubilization of zinc oxide and phosphate was accompanied by an increase in the H+ concentration of the medium, probably a consequence of the production of 2-ketogluconic acid.

Kulak Burun Bogaz Ihtis Derg, 2002 Mar-Apr, 9(2), 106 - 11
{Efficacy of topical ciprofloxacin and tobramycin in combination with dexamethasone in the treatment of chronic suppurative otitis media}; Kaygusuz I et al.; OBJECTIVES: To evaluate the efficacy of topical ciprofloxacin and tobramycin with and without topical dexamethasone in the treatment of chronic suppurative otitis media without cholesteatoma . PATIENTS AND METHODS: The study included 103 ears of 80 patients (49 males, 31 females; mean age 31 years; range 18 to 60 years) with chronic suppurative otitis media without cholesteatoma . The patients were randomly divided into four groups to receive topical applications of either ciprofloxacin and tobramycin alone, or in combination with dexamethasone . Cultures were obtained from the ears preoperatively and 24 hours after treatment . RESULTS: Aerobic bacteria were isolated in 94.1% of patients before the treatment, the most common being Pseudomonas aeruginosa (38.9%) . With dexamethasone, the clinical response for ciprofloxacin and tobramycin increased from 80% to 90% and from 70% to 75%, respectively, but this improvement was not significant (p > 0.03) . Addition of dexamethasone to ciprofloxacin decreased the recovery period from 14 days to seven days, whereas no change (7 days) was observed with tobramycin . CONCLUSION: The efficacy of ciprofloxacin and tobramycin were similar in the treatment of chronic suppurative otitis media . Addition of dexamethasone to ciprofloxacin decreased the treatment period.

Antimicrob Agents Chemother, 2002 Aug, 46(8), 2696 - 9
Extrusion of penem antibiotics by multicomponent efflux systems MexAB-OprM, MexCD-OprJ, and MexXY-OprM of Pseudomonas aeruginosa; Okamoto K et al.; The high intrinsic penem resistance of Pseudomonas aeruginosa is due to the interplay among the outer membrane barrier, the active efflux system MexAB-OprM, and AmpC beta-lactamase . We studied the roles of two other efflux systems, MexCD-OprJ and MexXY-OprM, in penem resistance by overexpressing each system in an AmpC- and MexAB-OprM-deficient background and found that MexAB-OprM is the most important among the three efflux systems for extrusion of penems from the cell interior.

Antimicrob Agents Chemother, 2002 Aug, 46(8), 2575 - 81
Ciprofloxacin in polyethylene glycol-coated liposomes: efficacy in rat models of acute or chronic Pseudomonas aeruginosa infection; Bakker-Woudenberg IA et al.; In a previous study in experimental Klebsiella pneumoniae pneumonia, the therapeutic potential of ciprofloxacin was significantly improved by encapsulation in polyethylene glycol-coated ("pegylated") long-circulating (STEALTH) liposomes . Pegylated liposomal ciprofloxacin in high doses was nontoxic and resulted in relatively high and sustained ciprofloxacin concentrations in blood and tissues, and hence an increase in the area under the plasma concentration-time curve (AUC) . These data correspond to data from animal and clinical studies showing that for fluoroquinolones the AUC/MIC ratio is associated with favorable outcome in serious infections . Clinical failures and the development of resistance are observed for marginally susceptible organisms like Pseudomonas aeruginosa and for which sufficient AUC/MIC ratios cannot be achieved . In the present study the therapeutic efficacy of pegylated liposomal ciprofloxacin was investigated in two rat models of Pseudomonas aeruginosa pneumonia . In the acute model pneumonia developed progressively, resulting in a rapid onset of septicemia and a high mortality rate . Ciprofloxacin twice daily for 7 days was not effective at doses at or below the maximum tolerated dose (MTD) . However, pegylated liposomal ciprofloxacin either at high dosage or given at low dosage in combination with free ciprofloxacin on the first day of treatment was fully effective (100% survival) . Obviously, prolonged concentrations of ciprofloxacin in blood prevented death of the animals due to early-stage septicemia in this acute infection . However, bacterial eradication from the left lung was not effected . In the chronic model, pneumonia was characterized by bacterial persistence in the lung without bacteremia, and no signs of morbidity or mortality were observed . Ciprofloxacin administered for 7 days at the MTD twice daily resulted in killing of more than 99% of bacteria in the lung; this result can also be achieved with pegylated liposomal ciprofloxacin given once daily . Complete bacterial eradication is never observed.

Antimicrob Agents Chemother, 2002 Aug, 46(8), 2400 - 8
Class 1 integron containing a new gene cassette, aadA10, associated with Tn1404 from R151; Partridge SR et al.; The carbenicillin, gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin resistance determinants found on Pseudomonas aeruginosa plasmid R151 have previously been shown to translocate to another plasmid, R388, and it was inferred that a transposon, Tn1404, carried the resistance determinants . Sequencing of the cassette array from the plasmid known as R388::Tn1404 revealed two known gene cassettes, oxa10 and aadB, and a previously unidentified cassette determining resistance to streptomycin and spectinomycin, here designated aadA10, in the order oxa10-aadB-aadA10 . These cassettes replaced the dfrB2-orfA cassette array of R388, indicating that movement of the resistance determinants from R151 to R388 resulted from recombinational exchange between two class 1 integrons rather than transposition . The AadA10 protein is most closely related to AadA6 (85% identical) and AadA7 (80% identical) . The aadA10 cassette found here has only a simple site containing a 7-bp spacer derived from attI1 in place of a 59-base element and is likely to represent a derivative of the complete cassette . IntI1-mediated deletion of the aadA10 cassette was not detected, indicating that this single simple site is either inactive or only weakly active.

Australas J Dermatol, 2002 Aug, 43(3), 202 - 6
Late-onset warfarin necrosis; Scarff CE et al.; A 43-year-old woman developed tenderness and induration of her thighs and lower abdomen, 56 days after commencing warfarin for aortic and mitral valve replacements . Investigations showed elevated inflammatory markers, mild renal impairment, normal echocardiogram and low protein C and S levels consistent with warfarin therapy . Three weeks later, purpuric areas evolved into large tender haemoserous bullae, which broke down to form ulcers . Histology confirmed the clinical impression of warfarin-induced skin necrosis with dermal and subcutaneous venular thrombi . Despite cessation of warfarin and commencement of heparin, the lesions progressed . When the patient became febrile, blood cultures grew Pseudomonas aeruginosa, which was treated with intravenous imipenem and vancomycin . Wound swabs grew methycillin-resistant Staphylococcus aureus and the antibiotics were changed . The patient developed septic shock and, despite intensive care management, her condition deteriorated and she died 9 weeks after the onset of the skin symptoms.

Immunol Cell Biol, 2002 Aug, 80(4), 340 - 5
1 alpha,25-Dihydroxyvitamin D3 inhibits pro-inflammatory cytokine and chemokine expression in human corneal epithelial cells colonized with Pseudomonas aeruginosa; Xue ML et al.; The cytokines IL-1 beta, IL-6 and the chemokine IL-8 are key mediators of host inflammation . 1 alpha,25-Dihydroxyvitamin D3 (VD3) has been shown to regulate host immune responses in vivo and in vitro . The purpose of this study was to investigate whether the addition of VD3 to human corneal epithelial cells colonized with Pseudomonas aeruginosa altered the expression of IL-1 beta, IL-6 and IL-8 . An immortalized human corneal epithelial (HCE) cell line was used in this study . After growth to confluency, HCE cells were challenged with P . aeruginosa strain 6294 in the presence or absence of 10-6 mol/L VD3 for 4 h, 8 h and 12 h . Gene expression of IL-1 beta, IL-6 and IL-8 was detected by reverse transcription-PCR (RT-PCR) from total RNA extracted from HCE cells . Protein concentrations of IL-1 beta, IL-6 and IL-8 in culture supernatants were measured by ELISA . Addition of VD3 to HCE cells colonized with P . aeruginosa significantly inhibited the expression of IL-1 beta and IL-8 mRNA and protein (P < 0.05) . Although the expression of IL-6 mRNA was stimulated at 12 h post-challenge (P < 0.05), the expression of IL-6 protein was inhibited at all time points after the addition of VD3 . In conclusion, this study demonstrated that VD3 inhibited the P . aeruginosa-induced expression of IL-1 beta, IL-6 and IL-8 in HCE cells, suggesting that this vitamin may have the potential to become a novel anti-inflammatory agent in ocular disease.

Immunol Cell Biol, 2002 Aug, 80(4), 323 - 7
Macrophage inflammatory protein-2 and vascular endothelial growth factor regulate corneal neovascularization induced by infection with Pseudomonas aeruginosa in mice; Xue ML et al.; Pseudomonas aeruginosa ocular infection causes extensive corneal neovascularization . The purpose of the present study was to investigate the role of the angiogenic factors macrophage inflammatory protein-2 (MIP-2) and vascular endothelial growth factor (VEGF) in the regulation of corneal neovascularization during P . aeruginosa ocular infection . After administering anti-MIP-2 antibody or control antibody, mouse corneas were challenged with P . aeruginosa . The expression of MIP-2 and VEGF was detected using an ELISA from ocular homogenates . Corneal neovascularization was examined by histology . The cellular sources of MIP-2 and VEGF were identified by immunohistochemistry . In addition, protein expression of MIP-2 and VEGF in isolated corneas was measured to determine the ability of the cornea to produce these two mediators . Results showed that the expression of MIP-2 and VEGF was significantly (P < 0.05) elevated after bacterial infection, and high levels of these two mediators paralleled the extensive corneal neovascularization seen at later stages of the infection . Anti-MIP-2 antibody treatment resulted in a significant (P < 0.05) reduction in VEGF expression and in corneal neovascularization . Both corneal resident cells and infiltrating neutrophils had the ability to produce MIP-2 and VEGF after stimulation . The present study demonstrates that both MIP-2 and VEGF are important mediators in the regulation of corneal neovascularization caused by P . aeruginosa infection, and that MIP-2 regulates the production of VEGF.

Chest, 2002 Jul, 122(1), 219 - 26
Pharmacokinetics and bioavailability of aerosolized tobramycin in cystic fibrosis; Geller DE et al.; STUDY OBJECTIVES: To describe the pharmacokinetics and bioavailability of inhaled tobramycin (TOBI; Chiron Corporation; Seattle, WA), 300-mg dose, delivered by a nebulizer (PARI LC Plus; Pari Respiratory; Richmond, VA) and a compressor (Pulmo-Aide, model 5650D; DeVilbiss Health Care; Somerset, PA) in cystic fibrosis (CF) patients during the pivotal phase III trials . DESIGN: Data from two identical, 24-week, randomized, double-blind, placebo-controlled, parallel-group studies . SETTING: US sites randomized 258 patients with CF to receive tobramycin, 300 mg twice daily, in three 28-day on/28-day off treatment cycles . MEASUREMENT: Tobramycin sputum concentrations were assessed 10 min after the first and last doses were administered in the 20-week study . Serum tobramycin concentrations were assessed before and 1 h after the first and last doses had been administered . The population estimate of the apparent clearance was used to estimate the bioavailability fraction . RESULTS: The mean peak sputum concentration was 1,237 microg/g . About 95% of patients achieved sputum concentrations > 25 times the minimum inhibitory concentration of the Pseudomonas aeruginosa isolates . One hour after the dose, the mean serum concentration was 0.95 microg/mL . Tobramycin did not accumulate in the sputum or serum over the course of the study . Pharmacokinetic data were best represented by a two-compartment model with biexponential decay and slope estimates comparable to those following parenteral administration . The estimated systemic bioavailability after aerosol administration was 11.7% of the nominal dose . CONCLUSIONS: The administration of tobramycin, 300 mg bid, in a 28-day off/28-day on regimen produced low serum tobramycin concentrations, reducing the potential for systemic toxicity . High sputum concentrations ensure efficacious antibiotic levels at the site of the infection . Inhaled tobramycin significantly improved the therapeutic ratio over that of parenteral aminoglycosides.

FEMS Microbiol Lett, 2002 Jul 2, 212(2), 249 - 54
Efflux of chromate by Pseudomonas aeruginosa cells expressing the ChrA protein; Pimentel BE et al.; The ChrA protein of Pseudomonas aeruginosa plasmid pUM505 confers resistance to chromate . Using an in vitro system, we reported {Alvarez, A.H . et al . (1999) J . Bacteriol . 181, 7398-7400} that chromate resistance is based on energy-dependent efflux of chromate . It is shown here that ChrA determines in vivo efflux of 51CrO(4)(2-) as well . Chromate-loaded cell suspensions of P . aeruginosa strain PAO1 harboring recombinant plasmid pEPL1, which expresses the ChrA protein, showed accelerated efflux of 51CrO(4)(2-) as compared to the plasmidless chromate-sensitive derivative . After a 10-min loading, about 40% of 51CrO(4)(2-) was lost from resistant cells in 15 min . Chromate efflux by resistant cells showed a typical saturation kinetics with an apparent K(m) of 82+/-11 microM chromate and a V(max) of 0.133+/-0.009 nmol chromate min(-1) (mg protein)(-1) . Oxyanions sulfate and molybdate inhibited chromate efflux in a concentration-dependent fashion, whereas arsenate and ortho-vanadate had no significant effect on chromate release . Inhibition of chromate extrusion by valinomycin, nigericin, and carbonyl cyanide m-chlorophenylhydrazone, but not by oligomycin or dicyclohexylcarbodiimide, indicated that chromate efflux was driven by the membrane potential.

Eur J Ophthalmol, 2002 May-Jun, 12(3), 225 - 7
Epidemic endophthalmitis after cataract surgery; Ernest J et al.; PURPOSE: We analyzed the results of pars plana vitrectomy in group of patients with a view to establishing risk factors, optimal therapy, surgical technique and the best timing of the pars plana vitrectomy . METHODS: Eight patients presented features of bacterial endophthalmitis within two days of cataract extraction . We examined the relations between visual outcome and the identity of infecting species, optimal therapy, surgical technique and vitrectomy timing . RESULTS: Pseudomonas aeruginosa was a pathogenic microbe . Unfortunately the source of infection was not found . The long-term (9-12 months) results are not good . Final visual acuity of all eight patients oscillated between 20/200 and no light perception . Three patients were first treated with intravitreal ATB application and vitrectomy followed with 36 hours lateney . Their final VA was no light perception in two patients and hand motion in one . The outcome was better in five patients operated immediately after the onset of endophthalmitis . Final visual acuity in this group was between hand motion and 20/200 . CONCLUSIONS: Visual prognosis in cases of endophthalmitis is closely related to the type of infecting organism, the visual acuity at presentation, and the speed of progression of inflammatory signs . The need for prompt vitrectomy as the only chance of retaining at least basic visual functions is fully demonstrated.

Pediatr Pulmonol, 2002 Aug, 34(2), 91 - 100
Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis; Emerson J et al.; We conducted a registry-based study to determine prognostic indicators of 8-year mortality and morbidity in young children with cystic fibrosis (CF) . Patients ages 1-5 years from the 1990 U.S . Cystic Fibrosis Foundation (CFF) National Patient Registry served as the study cohort (N = 3,323) . Registry data provided information on baseline characteristics in 1990, 8-year mortality, and clinical outcomes in 1998.P . aeruginosa respiratory infection was found to be a major predictor of morbidity and mortality . The 8-year risk of death was 2.6 times higher in patients who had respiratory cultures positive for P . aeruginosa in 1990 (95% confidence interval 1.6, 4.1) than in children without P . aeruginosa in their respiratory cultures . Culture-positive patients in 1990 also had a significantly lower percent predicted forced expiratory volume in 1 sec (FEV(1)) and weight percentile at follow-up, and they had an increased risk of continued P . aeruginosa respiratory infection and hospitalization for acute respiratory exacerbation in 1998 . Among the other predictors of increased morbidity and mortality were lower baseline weight percentiles and number of CF-related hospitalizations during the baseline year.These findings confirm reports from previous smaller studies of outcomes among young children with CF, and highlight the potential to decrease the morbidity and mortality of young patients with CF through early intervention .

J Infect Chemother, 2002 Jun, 8(2), 138 - 44
CP6679, a new injectable cephalosporin with broad spectrum and potent activities against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa; Ida T et al.; The antibacterial activities of CP6679, a new injectable cephalosporin with a broad antibacterial spectrum, were compared with those of other cephalosporins . CP6679 had stronger in-vitro activity than ceftazidime and cefpirome against methicillin-resistant Staphylococcus aureus(MRSA), methicillin-resistant coagulase-negative staphylococci, and Pseudomonas aeruginosa . Its activity against MRSA was eight times stronger than that of cefpirome, and it showed high binding affinity for penicillin-binding protein 2' of MRSA . Furthermore, the antibacterial activity of CP6679 against ceftazidime-resistant and imipenem-resistant P . aeruginosawas eight times stronger than that of ceftazidime and four times stronger than that of imipenem . In addition to its in-vitro activities, CP6679 showed the highest efficacy among all cephalosporins tested in murine models of systemic infection induced by MRSA or P . aeruginosa . It was more effective than vancomycin and cefpirome against respiratory tract infections induced by MRSA in mice.

Eur Respir J, 2002 Jun, 19(6), 1136 - 41
Colistin stimulates the activity of neutrophil elastase and Pseudomonas aeruginosa elastase; Jones A et al.; Nonantimicrobial effects of antibiotics may contribute to their activity in the treatment of infective airway disease . The aim of this study was to identify antibiotics used for the treatment of infection in cystic fibrosis that may alter the activity of human neutrophil elastase (HNE) and Pseudomonas aeruginosa elastase (PE) . The effect of antibiotics on the activity of purified HNE and PE, and HNE in sputum was assessed using colourimetric and fluorescent substrate assays by kinetic measurements, and by examining the interaction of HNE with inhibitors . Ceftazidime, tobramycin, and gentamycin slightly inhibited purified HNE activity whereas erythromycin and colistin significantly stimulated purified HNE and PE (395 and 557%, respectively) . However, only colistin increased HNE activity in sputum (+102%) and was therefore studied in more detail . This increase in activity was not due an interference with the specific inhibition of HNE by alpha1-antitrypsin but colistin was found to reverse the inhibitory effects of small molecular weight molecules like heparin . Colistin increases the activity of human neutrophil elastase and Pseudomonas aeruginosa elastase, two proteases that contribute to the pathogenesis of cystic fibrosis airway disease.

Chemosphere, 2002 Jun, 47(9), 891 - 9
Biodegradability of aged pyrene and phenanthrene in a natural soil; Hwang S et al.; A study was conducted to evaluate the biodegradability of pyrene (PYR) and phenanthrene (PHE) aged in a natural soil . Both the single and binary systems were either biostimulated via a nutrient amendment or bioaugmented via an inoculation of the enriched bacteria and nutrients . Aging resulted in higher concentration of both compounds and smaller bacterial activity in the solution-phase . Surprisingly, the total biodegraded extent was greater in the aged soil system than in the freshly spiked system . As anticipated, biostimulation was not appropriate to attain an effective biodegradation in this study, and bioaugmentation achieved a substantial increase the total biodegradation extent . The above findings were attributed to indigenous Pseudomonas aeruginosa entering a stationary-phase during the 200-day aging and producing rhamnolipid biosurfactants . In addition, a different sampling technique (i.e., after vigorous hand-shaking) revealed a 15 times higher microbial population than the normal sampling from the stagnant solution . Therefore, PAH bioavailability in the aged soils can be underestimated when the microbial activity is determined only from the stagnant solution . Furthermore, cometabolism enhanced PYR degradation when PHE was present as a primary substrate.

J Bacteriol, 2002 Aug, 184(15), 4321 - 5
Mutant analysis shows that alanine racemases from Pseudomonas aeruginosa and Escherichia coli are dimeric; Strych U et al.; Alanine racemases are ubiquitous prokaryotic enzymes providing the essential peptidoglycan precursor D-alanine . We present evidence that the enzymes from Pseudomonas aeruginosa and Escherichia coli function exclusively as homodimers . Moreover, we demonstrate that expression of a K35A Y235A double mutation of dadX in E . coli suppresses bacterial growth in a dominant negative fashion.

J Bacteriol, 2002 Aug, 184(15), 4308 - 12
The mexR repressor of the mexAB-oprM multidrug efflux operon in Pseudomonas aeruginosa: characterization of mutations compromising activity; Adewoye L et al.; Mutations in mexR yield a multidrug resistance phenotype in nalB mutants of Pseudomonas aeruginosa as a result of derepression of the mexAB-oprM multidrug efflux operon . MexR produced by several nalB strains carried single amino acid changes that compromised MexR stability or its ability to dimerize . Changes at residues L95 and R21, however, produced a stable MexR protein capable of dimerization and, thus, likely compromised DNA binding.

J Bacteriol, 2002 Aug, 184(15), 4301 - 3
Glycine betaine transmethylase mutant of Pseudomonas aeruginosa; Serra AL et al.; The gene for glycine betaine transmethylase (gbt) was identified in Pseudomonas aeruginosa strain Fildes III by biochemical, physiological, and molecular approaches . Based on sequence analysis, the knockout gene corresponded to an open reading frame (ORF) named PA3082 in the genome of P . aeruginosa PAO1 . The translated product of this ORF displayed similarity to transferases of different microorganisms . Mutation in gbt blocked the utilization of choline and glycine betaine as carbon and nitrogen sources.

Diagn Microbiol Infect Dis, 2002 Jul, 43(3), 239 - 43
Geographic variations in activity of broad-spectrum beta-lactams against Pseudomonas aeruginosa: summary of the worldwide SENTRY Antimicrobial Surveillance Program (1997-2000); Jones RN et al.; With reports of increasing resistance to antimicrobial agents among Pseudomonas aeruginosa clinical isolates worldwide, the activities of cefepime and eight other broad-spectrum beta-lactams against 6969 isolates collected during 1997-2000 from the four regions of the SENTRY Antimicrobial Surveillance Program . P . aeruginosa isolates were tested by the reference broth microdilution method against nine beta-lactam antimicrobial agents (aztreonam, cefepime, ceftazidime, imipenem, meropenem, piperacillin +/- tazobactam, ticarcillin +/- clavulanate), three aminoglycosides (amikacin, gentamicin, tobramycin), and two fluoroquinolones (ciprofloxacin, levofloxacin) . The strains were contributed by more than 100 medical centers . National Committee for Clinical Laboratory Standards criteria were used to identify susceptible and resistant isolates . P . aeruginosa strains from Latin America were generally the most resistant to all classes of antimicrobials, compared with strains from other regions . The beta-lactams exhibited a wide range of potency, with carbapenems most active (meropenem, 80-91% susceptible; imipenem, 76-88% susceptible) . Piperacillin/tazobactam was the most active penicillin (77-80% susceptible), and cefepime (67-83% susceptible) had an average 2% (range, 0.7-3.5%) greater susceptibility rate than ceftazidime (66-80% susceptible) across all regions . The rank order of beta-lactam activity according to percent resistant isolates in North American P . aeruginosa strains was: meropenem (4.8% resistant) > cefepime (6.8%) > imipenem (8.6%) > piperacillin/tazobactam (10.3%) > piperacillin (12.9%) . Only 2.3% and 6.5% of isolates were resistant to amikacin or tobramycin, respectively, and nearly 16% of P . aeruginosa strains were resistant to ciprofloxacin . Compared with other geographic regions, strains of P . aeruginosa remain most susceptible in North America . In all regions, aminoglycosides in combination with carbapenems, cefepime, or piperacillin/tazobactam would provide more potential antipseudomonal activity than fluoroquinolone combinations for wide-spectrum empiric regimens.

Wound Repair Regen, 2002 May-Jun, 10(3), 141 - 51
Early healing events in a porcine model of contaminated wounds: effects of nanocrystalline silver on matrix metalloproteinases, cell apoptosis, and healing; Wright JB et al.; A porcine model of wound healing was employed to examine the impact of nanocrystalline silver-coated dressings on specific wound healing events . Full-thickness wounds were created on the backs of pigs, contaminated with an experimental inoculum containing Pseudomonas aeruginosa, Fusobacterium sp., and coagulase-negative staphylococci, and covered with dressing products either containing silver or not . Nanocrystalline silver-coated dressings promoted rapid wound healing, particularly during the first several days post-injury . Healing was characterized by rapid development of well vascularized granulation tissue that supported tissue grafting 4 days post-injury, unlike control dressed wounds . The proteolytic environment of wounds treated with nanocrystalline silver was characterized by reduced levels of matrix metalloproteinases . Matrix metalloproteinases have been shown to be present in chronic ulcers at abnormally high levels, as compared with acute wounds, and may contribute to the nonhealing nature of these wounds . Cellular apoptosis occurred at a higher frequency in the nanocrystalline silver-treated wounds than in wounds dressed with other products . The results suggest that nanocrystalline silver may play a role in altering or compressing the inflammatory events in wounds and facilitating the early phases of wound healing . These benefits are associated with reduced local matrix metalloproteinase levels and enhanced cellular apoptosis.

Arch Soc Esp Oftalmol, 2002 Jul, 77(7), 397 - 9
{Pseudomonas aeruginosa corneal abscess refractory to fluoroquinolones}; Diaz Valle D et al.; PURPOSE/METHOD: To report a clinical case of a 21-year old male with a severe corneal abscess due to Pseudomonas aeruginosa refractory to intensive treatment with topical Ciprofloxacin . RESULTS/CONCLUSIONS: A corneal scrapping performed after a wash-out period showed Pseudomonas aeruginosa specimens, which were not sensitive to any of the fluoroquinolones tested . A fortified topical treatment with Ceftazidime healed the corneal abscess . Nowadays, the possibility of resistance to fluoroquinolones should be kept in mind, and we recommend treatment guided by the results of a microbiological study in severe and sight-threatening cases.

Thorax, 2002 Jul, 57(7), 596 - 601
Relationship between nutritional status and lung function in cystic fibrosis: cross sectional and longitudinal analyses from the German CF quality assurance (CFQA) project; Steinkamp G et al.; BACKGROUND: The German cystic fibrosis (CF) quality assurance (CFQA) project is a patient registry for CF which was founded in 1995 . Relevant clinical and laboratory data, respiratory function test results, complications, and CF treatments are entered into the database once a year for each patient . Using the database, a study was undertaken to elucidate the relationship between nutrition and lung function in a large patient cohort by cross sectional and longitudinal analysis . METHODS: A cohort of 3298 patients above 2 years of age was analysed . Patients were grouped according to the presence or absence of malnutrition (wasting and/or stunting) . Cross sectional and longitudinal analyses over 2 and 3 years including mixed model analyses were performed . RESULTS: The prevalence of abnormal weight for height (<90% predicted) increased with age from 19% in children aged <6 years to 38% in adults with CF . Patients with malnutrition had significantly lower mean values of vital capacity, arterial oxygen tension (PO(2)), and forced expiratory volume in 1 second (FEV(1)) and higher serum IgG (p<0.05) . Pseudomonas aeruginosa infection was also associated with decreased pulmonary function . Malnourished adolescents aged 12-18 years experienced a serious decline in FEV(1) of about 20% predicted, whereas mean FEV(1) values remained stable at above 80% predicted in adolescents of normal weight . Longitudinal follow up showed that malnourished patients of all ages and those with P aeruginosa infection had significantly worse lung function than their normally nourished counterparts and a greater yearly loss of FEV(1) % predicted . During 1 year of observation adolescents who experienced a >5% predicted decrease in weight for height had a concomitant mean loss of FEV(1) of 16.5% predicted during that year, whereas patients who gained relative weight had a parallel increase in FEV(1) of 2.1% predicted . CONCLUSIONS: These data emphasise the close relationship between nutrition, lung function, and clinical course in CF . Normal body weight and absence of P aeruginosa infection was associated with better preservation of lung function.

J Antimicrob Chemother, 2002 Jul, 50(1), 59 - 66
Macrolide-treated Pseudomonas aeruginosa induces paradoxical host responses in the lungs of mice and a high mortality rate; Kobayashi T et al.; OBJECTIVE: Accumulating data have demonstrated that macrolide antibiotics suppress Pseudomonas aeruginosa virulence, which may explain the efficacy of macrolides in clinical settings . We examined the virulence of macrolide-treated bacteria in vivo . METHODS: P . aeruginosa PAO-1 was grown for 24 h on agar containing sub-MIC antibiotics, and then mice were challenged intranasally with 10(7) cfu of bacteria . RESULTS AND CONCLUSIONS: The mortality rate of mice inoculated with bacteria grown in the presence of clarithromycin (10 mg/L), erythromycin (10 mg/L) or azithromycin (5 mg/L) was 80%, 80% and 100%, respectively . In contrast, none of the mice inoculated with non-treated bacteria or bacteria treated with other antibiotics died . Lung weight and protein concentration in bronchoalveolar lavage fluid (BALF) were significantly higher in the clarithromycin group between 3 and 9 h . Moreover, we detected higher levels of tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in the BALF of these mice . These data demonstrate that macrolide-treated P . aeruginosa induced paradoxically strong responses, such as elevation of TNF-alpha, NO and permeability in the lungs.

J Antimicrob Chemother, 2002 Jul, 50(1), 11 - 8
Identification of PSE and OXA beta-lactamase genes in Pseudomonas aeruginosa using PCR-restriction fragment length polymorphism; Bert F et al.; OBJECTIVE: A method using PCR-restriction fragment length polymorphism was developed to identify Pseudomonas aeruginosa beta-lactamase genes . METHODS: Two hundred and fifty-nine P . aeruginosa isolates were screened by PCR with 11 primer pairs designed to detect genes encoding PSE, OXA, TEM and SHV enzymes . PSE and OXA gene variants were distinguished by restriction of PCR products with endonucleases recognizing sites involved in point mutations . Nucleotide sequences were verified for a few isolates by sequencing the PCR products . RESULTS: Four isolates produced extended-spectrum beta-lactamases (ESBLs) according to the double disc synergy test . PCR detecting bla(PSE) genes was positive in 162 (62.5%) isolates: 151 carried bla(PSE-1) and 11 carried a variant encoding an enzyme differing from PSE-1 by a single amino acid substitution (Pro102 to Ser) . PCR detecting sequences for enzymes of the OXA-10 group was positive in 68 (26.3%) isolates: 31 carried bla(OXA-10), one carried bla(OXA-14) and 36 carried a new variant intermediate between bla(OXA-13) and bla(OXA-19) . The bla(OXA-2) gene was identified in 13 (5%) isolates . Two other isolates carried bla(OXA-2) variants encoding ESBLs differing from OXA-2 by a single amino acid substitution (Asp150 to Tyr and Trp159 to Cys, respectively) . PCR detecting sequences for enzymes of the OXA-1 group was positive in 12 (4.6%) isolates . A bla(TEM) gene was identified in five (1.9%) isolates (three bla(TEM-1), one bla(TEM-2), one bla(TEM-4)) . CONCLUSION: This approach is effective for screening P . aeruginosa for beta-lactamase gene carriage in epidemiological studies and for detecting new variants.

Emerg Infect Dis, 2002 Jul, 8(7), 697 - 701
Temporal changes in prevalence of antimicrobial resistance in 23 US hospitals; Fridkin SK et al.; Antimicrobial resistance is increasing in nearly all health-care-associated pathogens . We examined changes in resistance prevalence during 1996-1999 in 23 hospitals by using two statistical methods . When the traditional chi-square test of pooled mean resistance prevalence was used, most organisms appear to have increased in prevalence . However, when a more conservative test that accounts for changes within individual hospitals was used, significant increases in prevalence of resistance were consistently observed only for oxacillin-resistant Staphylococcus aureus, ciprofloxacin-resistant Pseudomonas aeruginosa, and ciprofloxacin- or ofloxacin-resistant Escherichia coli . These increases were significant only in isolates from patients outside intensive-care units (ICU) . The increases seen are of concern; differences in factors present outside ICUs, such as excessive quinolone use or inadequate infection-control practices, may explain the observed trends.

J Exp Med, 2002 Jul 1, 196(1), 109 - 18
Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa; Hirakata Y et al.; Pseudomonas aeruginosa is an important opportunistic human pathogen . Certain strains can transmigrate across epithelial cells, and their invasive phenotype is correlated with capacity to cause invasive human disease and fatal septicemia in mice . Four multidrug efflux systems have been described in P . aeruginosa, however, their contribution to virulence is unclear . To clarify the role of efflux systems in invasiveness, P . aeruginosa PAO1 wild-type (WT) and its efflux mutants were evaluated in a Madin-Darby canine kidney (MDCK) epithelial cell monolayer system and in a murine model of endogenous septicemia . All efflux mutants except a deltamexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT . In particular, a deltamexAB-oprM deletion strain was compromised in its capacity to invade or transmigrate across MDCK cells, and could not kill mice, in contrast to WT which was highly invasive (P < 0.0006) and caused fatal infection (P < 0.0001) . The other mutants, including deltamexB and deltamexXY mutants, were intermediate between WT and the deltamexAB-oprM mutant in invasiveness and murine virulence . Invasiveness was restored to the deltamexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT . We conclude that the P . aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.

Invest Ophthalmol Vis Sci, 2002 Jul, 43(7), 2278 - 84
Regulation of endotoxin-induced keratitis by PECAM-1, MIP-2, and toll-like receptor 4; Khatri S et al.; PURPOSE: Bacterial lipopolysaccharide (LPS, endotoxin) is a potent stimulator of inflammatory responses and is likely to contribute to microbial keratitis and to the pathogenesis of sterile corneal ulcers . The purpose of the present study was to identify specific mediators of endotoxin-induced keratitis . METHODS: The corneal epithelium of BALB/c, C3H/HeJ, and C3H/HeN mice was abraded, and Pseudomonas aeruginosa endotoxin (10 microg in 1 microL) was added . Stromal thickness and haze were measured by in vivo scanning confocal microscopy, and neutrophil recruitment determined by immunohistochemistry . RESULTS: Pseudomonas endotoxin induced a significant increase in stromal thickness and haze compared with untreated control corneas at each time point examined, and the severity coincided with neutrophil infiltration into the corneal stroma . Furthermore, systemic depletion of neutrophils completely abrogated endotoxin-induced increases in stromal thickness and haze, indicating an essential role for these cells . Expression of platelet endothelial cell adhesion molecule (PECAM)-1 on vascular endothelium and production of macrophage inflammatory protein (MIP)-2 in the corneal stroma were also significantly elevated after exposure to endotoxin, and antibody blockade inhibited neutrophil recruitment to the cornea and abrogated endotoxin-induced increases in stromal thickness and haze . In LPS-hyporesponsive C3H/HeJ mice, PECAM-1 and MIP-2 were not upregulated after exposure to endotoxin, and endotoxin-induced keratitis did not develop in these mice . CONCLUSIONS: These findings demonstrate that endotoxin-induced keratitis is regulated by toll-like receptor-4 (TLR4)-dependent expression of PECAM-1 and MIP-2, which are essential for recruitment of neutrophils to this site and for development of endotoxin-induced stromal disease.

Biochim Biophys Acta, 2002 Jul 3, 1571(3), 190 - 200
IMP-1 metallo-beta-lactamase: effect of chelators and assessment of metal requirement by electrospray mass spectrometry; Siemann S et al.; Metallo-beta-lactamases have attracted considerable attention due to their role in microbial resistance to beta-lactam antibiotics . IMP-1, the binuclear Zn-dependent beta-lactamase produced by Pseudomonas aeruginosa and other microorganisms, is of particular interest in view of its increasing prevalence . An examination of the susceptibility of IMP-1 to inactivation by six different divalent metal ion chelators has revealed that all except Zincon cause inhibition by forming a complex with the holoenzyme . Exposure of the enzyme to dipicolinic acid (DPA), the most potent inhibitor, results in the production of the mononuclear Zn form of the protein as determined by electrospray ionization mass spectrometry (ESI-MS) under nondenaturing conditions . This mononuclear Zn species was found to be catalytically competent . Studies with the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) show that the two zinc centers in IMP-1 differ in their accessibility, a feature that could be overcome in the presence of guanidine hydrochloride (GdnHCl, 1.5 M).

Arch Intern Med, 2002 Jul 8, 162(13), 1483 - 92
The hospital water supply as a source of nosocomial infections: a plea for action; Anaissie EJ et al.; BACKGROUND: Microbiologically contaminated drinking water is a cause of community-acquired infection, and guidelines for prevention of such infections have been established . Microbes in hospital water can also cause nosocomial infection, yet guidelines for preventing such infections do not exist . The purpose of this review is to assess the magnitude of the problem caused by waterborne nosocomial infections and to plea for immediate action for their prevention . METHODS: We conducted a MEDLINE search of the literature published between January 1, 1966, and December 31, 2001 . STUDY SELECTION AND DATA EXTRACTION: Investigations in which microorganisms (other than Legionella species) caused waterborne nosocomial infections and public health agency recommendations for drinking water . RESULTS: Forty-three outbreaks of waterborne nosocomial infections have been reported, and an estimated 1400 deaths occur each year in the United States as a result of waterborne nosocomial pneumonias caused by Pseudomonas aeruginosa alone . Despite the availability of effective control measures, no clear guidelines exist for the prevention of these infections . By contrast, guidelines for the prevention of community-acquired waterborne infections are now routinely used . Hospitals caring for patients at high risk for infection do not enforce the standards of water quality recommended by US and United Kingdom public health agencies for the patients' community counterparts . CONCLUSION: Because of the seriousness of these nosocomial waterborne infections and the availability, low cost, and proven effectiveness of sterile water, we recommend that hospitalized patients at high risk for infection avoid exposure to hospital water and use sterile water instead.

J Clin Microbiol, 2002 Jul, 40(7), 2420 - 4
Occurrence of a multidrug-resistant Pseudomonas aeruginosa clone in different hospitals in Rio de Janeiro, Brazil; Pellegrino FL et al.; Multidrug-resistant Pseudomonas aeruginosa nosocomial infections are increasingly recognized worldwide . The existence of metallo-beta-lactamase- and extended-spectrum beta-lactamase-producing isolates exhibiting resistance to most beta-lactam antimicrobial agents greatly complicates the clinical management of patients infected with such isolates . Since 1998, P . aeruginosa isolates resistant to all commercially available antimicrobial agents have been detected at a university-affiliated public hospital in Rio de Janeiro, Brazil . The present study was designed to characterize the antimicrobial resistance profiles and the genetic diversity of the P . aeruginosa strains isolated at this hospital and four private hospitals in Rio de Janeiro . Between April 1999 and March 2000, 200 consecutive isolates were obtained and analyzed for antimicrobial resistance . The genetic diversity of a selected number of them was evaluated by pulsed-field gel electrophoresis and PCR with the ERIC-2 primer . A predominant genotype, designated genotype A, was identified among isolates from four of the five hospitals evaluated . Eighty-four ceftazidime-resistant isolates were evaluated for metallo-beta-lactamase production, which was detected in 20 (91%) of 22 genotype A isolates and 11 (18%) of 62 isolates belonging to other genotypes (P < 0.05) . Two metallo-beta-lactamase-producing genotype A isolates also produced an extended-spectrum beta-lactamase . The occurrence of multidrug-resistant P . aeruginosa strains belonging to a unique genotype in different hospitals in Rio de Janeiro underscores the importance of the contribution of a single clone to the increase in the incidence of multidrug-resistant P . aeruginosa nosocomial infections.

J Biol Chem, 2002 Sep 20, 277(38), 35489 - 95 Epub 2002 Jun 27.
Involvement of toll-like receptor (TLR) 2 and TLR4 in cell activation by mannuronic acid polymers; Flo TH et al.; The alginate capsule produced by the human pathogen Pseudomonas aeruginosa is composed mainly of mannuronic acid polymers (poly-M) that have immunostimulating properties . Poly-M shares with lipopolysaccharide the ability to stimulate cytokine production from human monocytes in a CD14-dependent manner . In the present study we examined the role of Toll-like receptor (TLR) 2 and TLR4 in responses to poly-M . Blocking antibodies to TLR2 and TLR4 partly inhibited tumor necrosis factor production induced by poly-M in human monocytes, and further inhibition was obtained by combining the antibodies . By transiently transfecting HEK293 cells, we found that membrane CD14 together with either TLR2 or TLR4/MD-2 could mediate activation by poly-M . Transfection of HEK293 cells with TLR2 and fluorescently labeled TLR4 followed by co-patching of TLR2 with an antibody revealed no association of these molecules on the plasma membrane . However, macrophages from the Tlr4 mutant C3H/HeJ mice and TLR4 knockout mice were completely non-responsive to poly-M, whereas the tumor necrosis factor release from TLR2 knockout macrophages was half of that seen with wild type cells . Taken together the results suggest that both TLR2 and TLR4 are involved in cell activation by poly-M and that TLR4 may be required in primary murine macrophages.

J Med Chem, 2002 Jul 4, 45(14), 3112 - 29
Potent, novel in vitro inhibitors of the Pseudomonas aeruginosa deacetylase LpxC; Kline T et al.; Deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by LpxC is the first committed step in the Pseudomonas aeruginosa biosynthetic pathway to lipid A; homologous enzymes are found widely among Gram-negative bacteria . As an essential enzyme for which no inhibitors have yet been reported, the P . aeruginosa LpxC represents a highly attractive target for a novel antibacterial drug . We synthesized several focused small-molecule libraries, each composed of a variable aromatic ring, one of four heterocyclic/spacer moieties, and a hydroxamic acid and evaluated the LpxC inhibition of these compounds against purified P . aeruginosa enzyme . To ensure that the in vitro assay would be as physiologically relevant as possible, we synthesized a tritiated form of the specific P . aeruginosa glycolipid substrate and measured directly the enzymatically released acetate . Several of our novel compounds, predominantly those having fluorinated substituents on the aromatic ring and an oxazoline as the heterocyclic moiety, demonstrated in vitro IC(50) values less than 1 microM . We now report the synthesis and in vitro evaluation of these P . aeruginosa LpxC inhibitors.

Surg Endosc . 2002 Nov;16(11):1638 . Epub 2002 Jun 27.
Pseudomonas aeruginosa liver abscesses after diagnostic endoscopic retrograde cholangiography in two patients with sphincter of Oddi dysfunction type 2; Katsinelos P et al.; Patients with sphincter of Oddi dysfunction have a significantly increased rate of pancreatitis after manometry or sphincterotomy, but septic complications after diagnostic endoscopic retrograde cholangiopancreatography (ERCP) in patients with sphincter of Oddi dysfunction type 2 have not been reported . We describe two patients with sphincter of Oddi dysfunction type 2 in whom Pseudomonas aeruginosa serotype 10 septicemia and multiple small hepatic abscesses developed, all within 48 h after they underwent diagnostic ERCP . The sepsis and hepatic abscesses resolved after successful intravenous antibiotic administration . Despite scrupulous examination of the duodenoscope washing machine and the bottle of water, the bacteria responsible for the sepsis could not be isolated . It is possible that despite disinfection, a nondetectable colony of P . aeruginosa remained in a part of duodenoscope and proliferated to reach a potentially hazardous level the following day . This report highlights the importance administering antibiotic prophylaxis to patients with sphincter Oddi dysfunction type 2 who undergo ERCP, despite the functional nature of the disease.

J Bacteriol, 2002 Jul, 184(14), 3765 - 73
Functional analysis and regulation of the divergent spuABCDEFGH-spuI operons for polyamine uptake and utilization in Pseudomonas aeruginosa PAO1; Lu CD et al.; A multiple-gene locus for polyamine uptake and utilization was discovered in Pseudomonas aeruginosa PAO1 . This locus contained nine genes designated spuABCDEFGHI (spu for spermidine and putrescine utilization) . The physiological functions of the spu genes in utilization of two polyamines (putrescine and spermidine) were analyzed by using Tn5 transposon-mediated spu knockout mutants . Growth and uptake experiments support that the spuDEFGH genes specify components of a major ABC-type transport system for spermidine uptake, and enzymatic measurements indicated that spuC encodes putrescine aminotransferase with pyruvate as the amino group receptor . Although spuA and spuB mutants showed an apparent defect in spermidine utilization, the biochemical functions of the gene products have yet to be elucidated . Assays of lacZ fusions demonstrated the presence of agmatine-, putrescine-, and spermidine-inducible promoters for the spuABCDEFGH operon and the divergently transcribed spuI gene of unknown function . Since the observed induction effect of agmatine was abolished in an aguA mutant where conversion of agmatine into putrescine was blocked, putrescine or spermidine, but not agmatine, serves as the inducer molecule of the spuA-spuI divergent promoters . S1 nuclease mappings confirmed further the induction effects of the polyamines on transcription of the divergent promoters and localized the transcription initiation sites . Gel retardation assays with extracts from the cells grown on putrescine or spermidine demonstrated the presence of a polyamine-responsive regulatory protein interacting with the divergent promoter region . Finally, the absence of the putrescine-inducible spuA expression and putrescine aminotransferase (spuC) formation in the cbrB mutant indicated that the spu operons are regulated by the global CbrAB two-component system perhaps via the putative polyamine-responsive transcriptional activator.

Artif Organs, 2002 Jul, 26(7), 636 - 46
An infection-preventing bilayered collagen membrane containing antibiotic-loaded hyaluronan microparticles: physical and biological properties; Lee JE et al.; An infection-preventing bilayered membrane consisting of a dense and porous collagen membrane has been developed . The membrane was fabricated using a combined freeze-drying/air-drying method . Hyaluronan (HA) microparticles containing silver sulfadiazine (AgSD) were fabricated by gelling an HA solution with calcium chloride and were incorporated into collagen layers to allow the sustained release of AgSD . In vitro biodegradability of the membrane and the release of AgSD from the membrane could be controlled by cross-linking the membrane with ultraviolet (UV) irradiation . In a cytotoxicity test, cellular damage was minimized by the sustained release of AgSD from dressings . The antibacterial capacity of the material against Pseudomonas aeruginosa was investigated using the Bauer-Kirby disk diffusion test, and bacterial growth was found to be inhibited for 4 days . In vivo tests showed that the bilayered membrane was associated with greater tissue regeneration than a polymeric membrane and with no infection-related biological signs.

Biochemistry, 2002 Jul 2, 41(26), 8438 - 46
Role of protein flexibility in the catalytic cycle of p-hydroxybenzoate hydroxylase elucidated by the Pro293Ser mutant; Palfey BA et al.; Proline 293 of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa is in a highly conserved region of the flavoprotein aromatic hydroxylases . It is thought to impart rigidity to the backbone, as it partially cradles the FAD in these hydroxylases . Thus, this residue has been substituted with serine by site-directed mutagenesis to investigate the importance of flexibility of the peptide segment in catalysis . Differential scanning calorimetry demonstrated that the mutation has decreased the stability of the folded mutant protein compared to the wild-type PHBH . The increased flexibility in the protein backbone enhanced the accessibility of the flavin hydroperoxide intermediate to the solvent, causing an increase in the elimination of H(2)O(2) from this labile intermediate and, consequently, a decrease in the efficiency of substrate hydroxylation . Additionally, the increased accessibility of this mutant form of the enzyme makes it more susceptible than the wild-type enzyme to being trapped in the hydroxyflavin intermediate form in the presence of high levels of p-hydroxybenzoate . The mutation also lowers the pK(a) of the phenolic oxygen of bound p-hydroxybenzoate, and eliminates the pH dependence of the rate constant for flavin reduction by NADPH . These experimental observations lead to a model that explains how the wild-type protein can sense the charge of the 4-substituent of the aromatic ligand and link this charge to a flavin conformational change that is required for reaction with NADPH: (i) The peptide oxygen of Pro 293 is repelled by the negative charge of the phenolic oxygen of p-hydroxybenzoate . (ii) This repulsion is transmitted through the peptide backbone, causing the movement of Asn 300 . (iii) The change in the position of Asn 300 triggers the movement of the flavin from the largely buried "in" conformation to the exposed, reactive "out" conformation.

South Med J, 2002 Jun, 95(6), 653 - 6
Person-to-person transmission of Pseudomonas pneumonia in the community: documentation by pulsed-field electrophoresis; Patel P et al.; Pseudomonas aeruginosa is a rare cause of community-acquired infection . The source of this organism has usually been inapparent or environmental (ie, contaminated humidifiers) . We documented transmission of P aeruginosa leading to cavitary pneumonia and lung abscess from daughter to mother and confirmed the clonal identity of our two patients' isolates using pulsed-field electrophoresis.

J Biol Chem, 2002 Sep 6, 277(36), 33334 - 7 Epub 2002 Jun 21.
Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases; Park KW et al.; The bacterium, Vitreoscilla, can induce the synthesis of a homodimeric hemoglobin under hypoxic conditions . Expression of VHb in heterologous bacteria often enhances growth and increases yields of recombinant proteins and production of antibiotics, especially under oxygen-limiting conditions . There is evidence that VHb interacts with bacterial respiratory membranes and cytochrome bo proteoliposomes . We have examined whether there are binding sites for VHb on the cytochrome, using the yeast two-hybrid system with VHb as the bait and testing every Vitreoscilla cytochrome bo subunit as well as the soluble domains of subunits I and II . A significant interaction was observed only between VHb and intact subunit I . We further examined whether there are binding sites for VHb on cytochrome bo from Escherichia coli and Pseudomonas aeruginosa, two organisms in which stimulatory effects of VHb have been observed . Again, in both cases a significant interaction was observed only between VHb and subunit I . Because subunit I contains the binuclear center where oxygen is reduced to water, these data support the function proposed for VHb of providing oxygen directly to the terminal oxidase; it may also explain its positive effects in Vitreoscilla as well as in heterologous organisms.

J Mol Biol, 2002 Jul 5, 320(2), 237 - 47
Structure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism; Frere F et al.; All natural tetrapyrroles, including hemes, chlorophylls and vitamin B12, share porphobilinogen (PBG) as a common precursor . Porphobilinogen synthase (PBGS) synthesizes PBG through the asymmetric condensation of two molecules of aminolevulinic acid (ALA) . Crystal structures of PBGS from various sources confirm the presence of two distinct binding sites for each ALA molecule, termed A and P . We have solved the structure of the active-site variant D139N of the Mg2+-dependent PBGS from Pseudomonas aeruginosa in complex with the inhibitor 5-fluorolevulinic acid at high resolution . Uniquely, full occupancy of both substrate binding sites each by a single substrate-like molecule was observed . Both inhibitor molecules are covalently bound to two conserved, active-site lysine residues, Lys205 and Lys260, through Schiff bases . The active site now also contains a monovalent cation that may critically enhance enzymatic activity . Based on these structural data, we postulate a catalytic mechanism for P . aeruginosa PBGS initiated by a C-C bond formation between A and P-side ALA, followed by the formation of the intersubstrate Schiff base yielding the product PBG.

FEMS Microbiol Lett, 2002 Jun 4, 211(2), 183 - 8
Photoinduced bactericidal activity against Pseudomonas aeruginosa by TiO(2) based thin films; Amezaga-Madrid P et al.; The photoinduced bactericidal capacity of TiO(2) based films was evaluated, using as model organism Pseudomonas aeruginosa . Thin films were obtained by spray pyrolysis; they included undoped, Cu doped, and Al doped TiO(2) . Scanning electron microscopy was used to observe the final effect of the irradiated films upon the bacteria . Depending on the composition and characteristics of the films, quantitative experiments show that bacterial inhibition varies between 28 and 96% . The order of magnitude of the average quantum yield of the films was determined between 10(-9) and 10(-11) inhibited bacterial per photon.

FEMS Microbiol Lett, 2002 Jun 18, 212(1), 101 - 6
Role of the Pseudomonas aeruginosa las and rhl quorum-sensing systems in rhlI regulation; de Kievit TR et al.; In Pseudomonas aeruginosa the LasR-LasI and RhlR-RhlI quorum-sensing (QS) systems control expression of numerous virulence genes in a population density-dependent fashion . In this study, we investigated regulation of the autoinducer synthase gene rhlI, which is responsible for C(4)-HSL signal production . Primer extension analysis was used to map the rhlI transcriptional start site and an upstream regulatory region was identified . Expression studies revealed that (i) this regulatory region is important for rhlI expression and (ii) although the rhl QS system will induce rhlI, las is the dominant regulator . Furthermore, we found that control of rhlI in Escherichia coli is markedly different than in P . aeruginosa.

Adv Microb Physiol, 2002, 46, 47 - 110
The extracytoplasmic function (ECF) sigma factors; Helmann JD; Bacterial sigma (sigma) factors are an essential component of RNA polymerase and determine promoter selectivity . The substitution of one sigma factor for another can redirect some or all of the RNA polymerase in a cell to activate the transcription of genes that would otherwise be silent . As a class, alternative sigma factors play key roles in coordinating gene transcription during various stress responses and during morphological development . The extracytoplasmic function (ECF) sigma factors are small regulatory proteins that are quite divergent in sequence relative to most other sigma factors . Many bacteria, particularly those with more complex genomes, contain multiple ECF sigma factors and these regulators often outnumber all other types of sigma factor combined . Examples include Bacillus subtilis (7 ECF sigma factors), Mycobacterium tuberculosis (10), Caulobacter crescentus (13), Pseudomonas aeruginosa (approximately 19), and Streptomyces coelicolor (approximately 50) . The roles and mechanisms of regulation for these various ECF sigma factors are largely unknown, but significant progress has been made in selected systems . As a general trend, most ECF sigma factors are cotranscribed with one or more negative regulators . Often, these include a transmembrane protein functioning as an anti-sigma factor that binds, and inhibits, the cognate sigma factor . Upon receiving a stimulus from the environment, the sigma factor is released and can bind to RNA polymerase to stimulate transcription . In many ways, these anti-sigma:sigma pairs are analogous to the more familiar two-component regulatory systems consisting of a transmembrane histidine protein kinase and a DNA-binding response regulator . Both are mechanisms of coordinating a cytoplasmic transcriptional response to signals perceived by protein domains external to the cell membrane . Here, I review current knowledge of some of the better characterized ECF sigma factors, discuss the variety of experimental approaches that have proven productive in defining the roles of ECF sigma factors, and present some unifying themes that are beginning to emerge as more systems are studied.

Harefuah, 2002 May, 141(5), 439 - 41, 498
{Home intravenous antibiotic therapy for osteomyelitis patients}; Steinmetz D et al.; OBJECTIVES: To evaluate both the safety and the costeffectiveness of the Home Intravenous Antibiotic Therapy (HIAT) program for the treatment of patients with osteomyelitis in the district of Haifa and Western Galilee . METHODS: We checked the medical records of all the patients with osteomyelitis who had been treated at home with intravenous antibiotics during 1999-2000 . We reviewed their records for etiological agents, types of antibiotics given, complications and cost evaluation . RESULTS: During the two year period (January 1999 to December 2000), 52 patients received 55 courses of HIAT . The total duration of treatments was 1187 days . Eighty-five percent of the patients were referred from clinical departments from one of the medical centers in our area . In 54.6% of cases the infecting agent is unknown . Among the known ones, Staphylococci and Pseudomonas aeruginosa were the most prevalent pathogens . Penicillins and Cephalosporins were the most common antimicrobial agent prescribed . The HIAT program saved NIS . 1,299,765 (approx . $365,000) during 1999-2000 . Only minor complications were present . CONCLUSIONS: HIAT for the treatment of patients with osteomyelitis is effective, safe and comfortable for the patients and has an important economic impact.

Artif Organs, 2002 Jun, 26(6), 512 - 20
The effect of apo-transferrin on bacterial adhesion to biomaterials; Ardehali R et al.; Apo-transferrin (apo-Tf), the iron deficient form of Tf, has been identified previously as a potent inhibitor of Staphylococcus epidermidis adhesion to polyurethane surfaces . In this study, the ability of apo-Tf to suppress the adhesion of two other strains of bacteria, namely a Gram-positive Staphylococcus aureus and a Gram-negative Pseudomonas aeruginosa to several biomaterials, including polystyrene, polymethylmethacrylate, and silicone, is documented . The presence of apo-Tf in the medium at 20 microg/ml lowered bacterial adhesion to all tested biomaterials more than fourfold . Moreover, apo-Tf exerted its inhibitory activity even when protein coated surfaces were used as substrates for bacterial adhesion . To emphasize the importance of apo-Tf in the prevention of bacterial adhesion, human serum was depleted of Tf, employing affinity chromatography, and was shown to lose its inhibitory activity toward bacterial adhesion . Upon addition of apo-Tf to Tf-depleted serum, the activity was reestablished, resulting in a marked reduction in the number of bacteria adhered to the surfaces . Following the enzymatic deglycosylation, apo-Tf retained its ability to prevent bacterial adhesion . These results indicate that the carbohydrate moiety does not seem to play a role in this activity . The presented data provide the evidence that the inhibitory activity of apo-Tf is not bacterial strain specific and that the presence of apo-Tf in the medium results in a significant reduction of bacterial adhesion to a variety of neat and/or protein coated surfaces.

Arch Dermatol, 2002 Jul, 138(7), 893 - 9
Necrotizing fasciitis: report of 39 pediatric cases; Fustes-Morales A et al.; BACKGROUND: Necrotizing fasciitis (NF) is a severe, life-threatening soft tissue infection . General features and risk factors for fatal outcome in children are not well known . OBJECTIVE: To characterize the features of NF in children and the risk factors for fatal outcome . DESIGN: Retrospective, comparative, observational, and longitudinal trial . SETTING: Dermatology department of a tertiary care pediatric hospital . PATIENTS: All patients with clinical and/or histopathological diagnosis of NF seen from January 1, 1971, through December 31, 2000 . MAIN OUTCOME VARIABLES: Incidence, age, sex, number and location of lesions, preexisting conditions, initiating factors, clinical and laboratory features, diagnosis at admission, treatment, evolution, sequelae, and risk factors for fatal outcome . RESULTS: We examined 39 patients with NF (0.018% of all hospitalized patients) . Twenty-one patients (54%) were boys . Mean age was 4.4 years . Single lesions were seen in 30 (77%) of patients, with 21(54%) in extremities . The most frequent preexisting condition was malnutrition in 14 patients (36%) . The most frequent initiating factor was varicella in 13 patients (33%) . Diagnosis of NF at admission was made in 11 patients (28%) . Bacterial isolations in 24 patients (62%) were polymicrobial in 17 (71%) . Pseudomonas aeruginosa was the most frequently isolated bacteria; gram-negative isolates, the most frequently associated bacteria . Complications were present in 33 patients (85%), mortality in 7 (18%), and sequelae in 29 (91%) of 32 surviving patients . The significant risk factor related to a fatal outcome was immunosuppression . CONCLUSIONS: Necrotizing fasciitis in children is frequently misdiagnosed, and several features differ from those of NF in adults . Immunosuppression was the main factor related to death . Early surgical debridement and antibiotics were the most important therapeutic measures.

Jpn J Antibiot, 2002 Apr, 55(2), 187 - 95
{Rapid bactericidal activities of carbapenems against Pseudomonas aeruginosa in simulating human plasma concentrations}; Ishii C et al.; The rapid bactericidal activities of panipenem (PAPM), imipenem (IPM), and meropenem (MEPM) against Pseudomonas aeruginosa were investigated by using in vitro pharmacodynamic model simulating the human plasma concentrations after intravenous drip infusion at 500 mg for 0.5 hours . Against P . aeruginosa PAO1, PAPM and IPM showed rapider reduction in viable cell counts than MEPM at 0.5 hours after exposure . All drugs showed more than 3 log10 reduction in viable cell counts at 2 hours after exposure and bacterial regrowth was not observed throughout 6 hours . The initial bactericidal activities of the drugs against 4 clinical isolates within 1 hour after exposure were also investigated by the same method . Against P . aeruginosa strain 12,475, the 3 drugs showed similar initial bactericidal activity but PAPM and IPM showed stronger initial bactericidal activity than MEPM against the other strains as did against P . aeruginosa PAO1 . The morphological change of a strain 12,489, for which the initial bactericidal activities were different largely, after 0.5 hours exposure to simulated drug-concentrations was investigated by scanning electron microscope . PAPM and IPM induced morphological changes in most of the cells and cell lysis and bulge formation . On the other hand, MEPM induced changes of the surface structure of cells and slightly elongated cells, but not cell lysis.

Jpn J Antibiot, 2002 Apr, 55(2), 181 - 6
{Antimicrobial activity of carbapenems and the combined effect with aminoglycoside against recent clinical isolates of Pseudomonas aeruginosa}; Tasaka K et al.; The carbapenem susceptibility of 32 strains of Pseudomonas aeruginosa recently isolated in Kakogawa municipal hospital was investigated . The MIC ranges of imipenem, panipenem, and meropenem were 0.25-16 mg/L, 0.5-16 mg/L, and < 0.03-4 mg/L, respectively, and meropenem showed the highest antipseudomonal activity among the three carbapenems tested . In the analysis based on the MIC interpretive standards established by NCCLS, the resistance rates of test strains for imipenem, panipenem, and meropenem were 6.3%, 15.6%, and 0%, respectively . We also investigated the in vitro combined effect of imipenem or meropenem with amikacin against another 20 isolates of P . aeruginosa by checkerboard titration assay . Antagonism (minimum FIC index > 2) was not observed in any combinations against all strains tested . Super-additive effects (minimum FIC index < 1) in the combination of imipenem and amikacin were observed in eight (40%) strains tested . In contrast, in the combination of meropenem and amikacin, super-additive effects were observed in 14 isolates (70%) . These results suggested that meropenem is superior to imipenem in combined effect with amikacin against P . aeruginosa . In conclusion, meropenem showed higher antipseudomonal activities than other carbapenems tested in both conditions, alone and in combination with amikacin . With regard to the clinical efficacy and prevention of antibiotic resistance, meropenem monotherapy or combination therapy with aminoglycoside is the most superior treatment for pseudomonal infections, and the findings in this study suggest that meropenem is still clinically very useful.

Commun Dis Public Health, 2002 Mar, 5(1), 23 - 6
Incidence of Pseudomonas aeruginosa in recreational and hydrotherapy pools; Moore JE et al.; Pseudomonas aeruginosa remains an important agent of opportunistic infection in patients, particularly those with respiratory complications and burns . One natural niche of this organism is water and water-associated facilities, hence the aim of this study was to examine specimens from recreational and hydrotherapy pools in Northern Ireland over a two-year period . Water specimens (n = 3,510) were obtained from three amenity categories, namely, 13 hydrotherapy pools (specimen number {n} = 323), 51 Jacuzzis/spas (n = 1,397) and 68 swimming pools (n = 1,790) . Specimens (100 ml) were filtered through a cellulose acetate (0.45 micron pore size) gridded filter and the membrane was placed on Pseudomonas CFC agar (Oxoid CM559 + SR103) and incubated at 37 degrees C for 48 +/- 2 h . Colonies that clearly showed pyocyanin production or met other identification criteria were considered P . aeruginosa . Of the amenities examined 4/13 hydrotherapy pools (30.8%), 37/51 Jacuzzis/spas (72.5%) and 26/68 swimming pools (38.2%) were positive for P . aeruginosa . The most heavily contaminated amenity category was the Jacuzzi/spa, where 34.7% and 12% of private and public sites respectively were positive for P . aeruginosa at a level of greater than 1,000 cfu 100 ml-1 . Approximately twice as many samples were positive in private Jacuzzis/spas compared to publicly operated facilities . There was a similar trend with respect to public and private hydrotherapy pools, though bacterial counts did not exceed 1,000 cfu 100 ml-1 . Recreational and therapeutic amenities involving the use of water may be a potential source of P . aeruginosa for susceptible patient groups, including patients with cystic fibrosis and bronchiectasis . This may vary depending on amenity type and public/private ownership of such amenities.

Antimicrob Agents Chemother, 2002 Jul, 46(7), 2169 - 73
Amino acid residues essential for function of the MexF efflux pump protein of Pseudomonas aeruginosa; Aires JR et al.; At least four broad-spectrum efflux pumps (Mex) are involved in elevated intrinsic antibiotic resistance as well as in acquired multidrug resistance in Pseudomonas aeruginosa . Substrate specificity of the Mex pumps has been shown to be determined by the cytoplasmic membrane component (MexB, MexD, MexF, and MexY) of the tripartite efflux pump system . Alignment of their amino acid sequences with those of the homologous AcrB and AcrD pump proteins of Escherichia coli showed conservation of five charged amino acid residues located in or next to transmembrane segments (TMS) . These residues were mutated in the MexF gene by site-directed mutagenesis and replaced by residues of opposite or neutral charge . MexF proteins containing combined D410A and A411G substitutions located in TMS4 were completely inactive . Similarly, the substitutions E417K (next to TMS4) and K951E (TMS10) also caused loss of activity towards all tested antibiotics . The substitution E349K in TMS2 resulted in a MexF mutant protein which was unable to transport trimethoprim and quinolones but retained partial activity for the transport of chloramphenicol . All mutated MexF proteins were expressed at comparable levels when tested by Western blot analysis . It is concluded that charged residues located in or close to TMS are essential for proper function of MexF.

J Vet Med B Infect Dis Vet Public Health, 2002 May, 49(4), 188 - 92
In vitro antimicrobial susceptibility of 183 Pseudomonas aeruginosa strains isolated from dogs to selected antipseudomonal agents; Seol B et al.; During the years from 1993 to 2000, 183 strains of Pseudomonas aeruginosa were isolated from different pathological specimens originating from dogs . Antimicrobial susceptibility patterns against 10 antipseudomonal agents were obtained on 183 P . aeruginosa strains . In vitro antimicrobial susceptibility testing was performed using the disk diffusion method (Kirby-Bauer) . Antimicrobial susceptibility profiles showed that among beta-lactam antibiotics, imipenem was the most active compound . Out of the 183 strains tested, 96.7% were sensitive to imipenem . Cefoperazone showed good in vitro activity against 86.9% of the tested strains . Against ceftazidime, 77.0% of strains showed sensitivity . An old penicillin, carbenicillin, gave only 71.6% sensitive strains . Sensitivity to amikacin was 87.4% and it was 83.1% to gentamicin . Pipimedic acid, a first-generation quinolone, was the least active compound of all those tested, 47.0% were resistant . The in vitro sensitivity against enrofloxacin showed that 71.0% strains were sensitive and 26.2% showed resistance . Almost all strains tested, 93.4%, were susceptible to ciprofloxacin and marbofloxacin . Besides imipenem, the quinolone antibiotics, marbofloxacin and ciprofloxacin were the most effective against P . aeruginosa strains isolated from dogs.

Scand J Infect Dis, 2002, 34(5), 372 - 8
Long-term antibiotic resistance surveillance of gram-negative pathogens suggests that temporal trends can be used as a resistance warning system; Sorberg M et al.; Antibiotic resistance among Gram-negative bacteria and antibiotic consumption were investigated at the Karolinska Hospital, Stockholm, Sweden over a 12-y period . The investigation showed an increase in ciprofloxacin resistance of Escherichia coli from 0% in 1991 to 7% in 1997 and to 11% in 1999 . Resistance among Pseudomonas aeruginosa isolates to ciprofloxacin increased from 2.5% in 1991 to 9.0% in 1997 and to 13% in 1999 . Resistance levels for norfloxacin showed the same high statistical significance in terms of the temporal trend . A more detailed analysis showed higher resistance against norfloxacin in specific wards . Relationships between antibiotic use and antibiotic susceptibility showed different patterns . The increased ciprofloxacin resistance of E . coli and P . aeruginosa during the study period was paralleled by an increased consumption of quinolones . During the 12-y study period the total use of cephalosporins increased 2.5-fold, while the levels of E . coli resistance to cefuroxime and cefotaxime remained stable . A third pattern was seen with trimethoprim-sulfamethoxazole, namely increasing resistance of E . coli as the use of trimethoprim-sulfamethoxazole declined . The analysis of resistance levels and antibiotic consumption in the present study suggests different mechanisms for the increased resistance . The significant trend of increased resistance to antibiotics over time constitutes an important warning system.

FEBS Lett, 2002 Jun 19, 521(1-3), 81 - 6
Halocidin: a new antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium; Jang WS et al.; From hemocytes of the tunicate Halocynthia aurantium we purified a new antimicrobial peptide named halocidin . The native peptide had a mass of 3443 Da and comprised two different subunits containing 18 amino acid residues (WLNALLHHGLNCAKGVLA) and 15 residues (ALLHHGLNCAKGVLA), which were linked covalently by a single cystine disulfide bond . Two different monomers were separately synthesized and used to make three additional isoforms (15 residue homodimer, 18 residue homodimer, heterodimer) . In antimicrobial assays performed with synthetic peptides of halocidin, it was confirmed that congeners of the 18 residue monomer were more active than those of the 15 residue monomer against methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa.

J Appl Microbiol, 2002, 93(1), 96 - 107
Adaptive resistance to benzalkonium chloride, amikacin and tobramycin: the effect on susceptibility to other antimicrobials; Joynson JA et al.; AIMS: To produce strains of antimicrobial-resistant Pseudomonas aeruginosa via adaptation to benzalkonium chloride, amikacin and tobramycin and to then examine the incidence, or otherwise, of cross-resistance between antibiotics and between antibiotics and benzalkonium chloride . METHODS AND RESULTS: Adaptation was obtained by progressive subculturing in subinhibitory concentrations of the antimicrobials . Pseudomonas aeruginosa NCIMB 10421 adapted to grow in high concentrations of benzalkonium chloride (BC) had lower MIC to antibiotics than the wild type, whereas Ps . aeruginosa adapted to grow in antibiotics had greater MIC to benzalkonium by a small degree . CONCLUSIONS: Adaptive resistance to BC of Ps . aeruginosa generally produced cultures with a decrease in resistance to several antibiotics . Adaptive resistance to the aminoglycosides Ak and Tm produced a low-level increase in tolerance to BC . The adaptive mechanisms of resistance appear to be different for the different types of antimicrobials used . SIGNIFICANCE AND IMPACT OF THE STUDY: The relationships between biocide and antibiotic resistance are complex . It appears, from this study, that an organism resistant to a common biocide can become sensitive to antibiotics, but the converse was not true . Could this observation be used in a strategy to alleviate antibiotic resistance?

J Asian Nat Prod Res, 2002 Jun, 4(2), 109 - 15
Ionone derivatives from Alternanthera sessilis; Ragasa CY et al.; The chloroform extract of the air-dried leaves of Alternanthera sessilis afforded a mixture of diastereomers of a new ionone derivative 1 whose structures were elucidated by extensive 1D and 2D NMR spectroscopy and mass spectrometry . Oxidative cleavage of 1 to aldehyde 3 with manganese dioxide confirmed diastereoisomerism arose from a racemic side chain chiral centre . Antimicrobial tests on the mixture of diastereomers and the derivative indicated that they have low activities against Pseudomonas aeruginosa and Trichophyton mentagrophytes.

Eur Rev Med Pharmacol Sci, 2001 Jul-Aug, 5(4), 131 - 8
Nebulized tobramycin in patients with chronic respiratory infections during clinical evolution of Wegener's granulomatosis; Terzano C et al.; Aminoglycosides are effective against Pseudomonas aeruginosa but with intravenous administration there are only very low concentrations achieved in sputum; therefore in order to obtain therapeutic levels in patients with endobronchial infections should be administered high doses with increased likelihood to produce both nephrotoxic and ototoxic effects . Direct aerosol delivery of aminoglycosides to the lower respiratory tract has the advantage to achieve high antibiotic sputum concentrations in the infected area with reduced risk of systemic toxic reactions because of minimal absorption into the circulation . Nowadays, except for patients suffering from cystic fibrosis and bronchiectasis, the administration of antibiotics through inhalers is not very much in use . The aim of this study was to administer nebulized tobramycin in chronic respiratory infections developed during the evolution of Wegener's Granulomatosis in order to obtain data concerning the safety and efficacy of inhaled aminoglycosides . The results obtained underlined an improvement in FEV1, FEF75 and PaO2 . The aerosolized tobramycin administered in 300 mg doses three times per day for four weeks, showed itself to be effective and safe, not causing any undesirable clinical or microbiological side-effects . Moreover, a long term treatment has been shown to control the Pseudomonas aeruginosa infection on the bronchial system in Wegener's granulomatosis and reduce the frequency of exacerbations in chronic patients.

Res Microbiol, 2002 May, 153(4), 221 - 6
The role of integrons in the dissemination of antibiotic resistance among clinical isolates of Pseudomonas aeruginosa from an intensive care unit in Brazil; Severino P et al.; Eighty-five Pseudomonas aeruginosa isolates resistant to various antibiotics were selected from the intensive care unit of a Brazilian hospital and analyzed for integron content by PCR . Fifty-four of them had class-1-related integrons, some of which were identical . Integron-positive isolates were statistically more resistant to aminoglicoside, quinolone and beta-lactam compounds . Ribotyping of integron-positive isolates demonstrated the presence of identical integrons among genetically unrelated strains in the hospital environment, confirming its role in the spread of gene cassettes in bacterial populations of clinical importance.

Infect Immun, 2002 Jul, 70(7), 3973 - 7
Activation of the Pseudomonas aeruginosa type III secretion system requires an intact pyruvate dehydrogenase aceAB operon; Dacheux D et al.; Pseudomonas aeruginosa clinical cystic fibrosis isolate CHA was mutagenized with Tn5Tc to identify new genes involved in type III secretion system (TTSS)-dependent cytotoxicity toward human polymorphonuclear neutrophils . Among 25 mutants affected in TTSS function, 14 contained the insertion at different positions in the aceAB operon encoding the PDH-E1 and -E2 subunits of pyruvate dehydrogenase . In PDH mutants, no transcriptional activation of TTSS genes in response to calcium depletion occurred . Expression in trans of ExsA restored TTSS function and cytotoxicity.

Chest, 2002 Jun, 121(6), 1754 - 60
Factors affecting the incidence of Stenotrophomonas maltophilia isolation in cystic fibrosis; Graff GR et al.; STUDY OBJECTIVES: To identify factors predisposing cystic fibrosis (CF) patients to Stenotrophomonas maltophilia infection and to determine whether coinfection with S maltophilia affects the clinical response to therapy with tobramycin solution for inhalation (TSI), 300 mg bid . DESIGN: Retrospective review of data collected from two identical, 6-month, randomized, placebo-controlled trials . SETTING: Sixty-nine CF centers in the United States . INTERVENTIONS: Active drug administration of 300 mg TSI . PATIENTS: Five hundred twenty CF patients with chronic Pseudomonas aeruginosa endobronchial infections . MEASUREMENTS AND RESULTS: A logistic regression analysis identified factors contributing to increased S maltophilia isolation frequency . In this multivariate analysis, the only significant predictors of S maltophilia isolation during the last month of the trial were the concomitant use of oral quinolones (primarily ciprofloxacin; p = 0.0015) and S maltophilia isolation prior to treatment (p < 0.0001) . Treatment group, gender, age, use of systemic or inhaled steroids, use of oral sulfonamide, IV cephalosporins, or penicillin antibiotics, baseline FEV(1) percent predicted, and pretreatment Aspergillus isolation were not significant predictors of subsequent S maltophilia infection . In addition, S maltophilia-positive culture frequency was compared to the change in pulmonary function . Patients who either never had culture results positive for S maltophilia or who were positive at <25% of observations had greater clinical response to TSI at the final study visit compared to patients who were positive at > or = 25% of observations . CONCLUSIONS: TSI therapy did not result in a greater risk for isolation of S maltophilia than standard care alone . In contrast, oral quinolone antibiotic use during the trial was associated with a 2.7-fold increased risk of having a culture positive for S maltophilia (p = 0.0015) . The use of TSI to suppress P aeruginosa resulted in improved lung function, regardless of S maltophilia culture frequency . However, improvement was not as great among patients who were persistently coinfected with S maltophilia.

Paediatr Respir Rev, 2002 Mar, 3(1), 82 - 8
Decisions facing the cystic fibrosis clinician at first isolation of Pseudomonas aeruginosa; Bush A; Chronic endobronchial infection with shape Pseudomonas aeruginosa in patients with cystic fibrosis is associated with more serious disease and reduced survival . Methods for reducing or preventing chronic infection with P . aeruginosa involve rigorous infection control measures and avoidance of cross-infection, which may include segregation of clinics according to microbiological status . The strains of shape P . aeruginosa first isolated from the lungs of cystic fibrosis patients are generally of a non-mucoid phenotype and sensitive to antibiotic therapy . There is some evidence that early aggressive antibiotic treatment may delay chronic infection, improve lung function and improve survival . Further research is needed into the accurate diagnosis of early infection with shape P . aeruginosa, which is often intermittent . In addition, the optimal treatment of patients at first isolation and early colonisation needs to be researched, including choice of antibiotic(s) and route, dosage and duration of antibiotic therapy.

Eur Arch Otorhinolaryngol, 2002 Apr, 259(4), 222 - 6
Reduction of neuronal and inducible nitric oxide synthase gene expression in patients with cystic fibrosis; Dotsch J et al.; As a consequence of diminished nitric oxide synthase (NOS) protein concentration, the airway concentration of nitric oxide (NO) is reduced in patients with cystic fibrosis (CF) . This appears to lead to a reduced elimination of such microorganisms as Pseudomonas aeruginosa . The objective of this study was to analyze whether inducible (iNOS), endothelial (eNOS) and neuronal (bNOS) NOS are reduced at mRNA level and if so whether this is caused directly by the defective CF transmembrane conductance regulator (CFTR) . Nasal polyps from three patients with CF and four otherwise healthy patients were obtained . The expression of the three NOS isoenzymes was quantified using real-time PCR . The iNOS expression was assessed in colon carcinoma cells (CaCo) transfected with a normal and a mutated (DeltaF508) CFTR . In CF patients, iNOS mRNA expression was 10-to 20-fold and bNOS gene expression was one-fifth to one-tenth that in control patients (P < 0.001) . In CaCo cells, iNOS gene expression under basal and endotoxin-stimulated conditions did not differ between cells transfected with a mutated CFTR and those transfected with an intact CFTR . This observation suggests that cystic fibrosis is associated with reduced iNOS and bNOS gene expression in nasopharyngeal tissue, possibly disturbing the barrier against infective agents already at the site of entrance.

Xenotransplantation, 2002 Jul, 9(4), 260 - 7
Lectin interactions with alpha-galactosylated xenoantigens; Kirkeby S et al.; alpha-Galactosylated xenoantigens (Galalpha1-3Galbeta1-4GlcNAcbeta1 and Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) are often detected with the alpha-Gal specific lectin Griffonia simplicifolia 1 isolectin B4 (GS1 B4) . However, this lectin exhibits a broad and variable specificity for carbohydrates terminating in alpha-Gal . Thus, both false positive and false negative results may occur when GS1 B4 is used to determine natural antigens in xeno (pig-to-primate) transplantation research . To refine the tools for detecting alpha-galactosylated antigens we have studied the binding of various alpha-galactophilic lectins to alpha-galactosylated neoglycoproteins . The lectins were: Euonymus europaeus agglutinin (EEA), Griffonia simplicifolia 1 isolectin B4 (GS1 B4), Maclura pomifera agglutinin (MPA) and Pseudomonas aeruginosa agglutinin (PA-IL) . Although both GS1 B4 and MPA strongly bound glycoconjugates terminating in Gal there seems to be some differentiation in their sugar binding preferences . MPA was the only lectin that showed high affinity for the pentasaccharide Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc and for the Galalpha-glycans on non-primate thyroglobulin . The length of the xenoantigenic carbohydrate chain may influence the nature of the inhibition when a simple sugar is used to inhibit GS1 B4 binding to the xenoantigen . Inhibition studies of MPA GS1 B4 interaction further suggest that both lectins attach to the same site of the carbohydrate antigen and that GS1 B4 in addition binds to at least one other site that has no affinity for MPA . When lectins are used for recognition and investigation of natural Galalpha-antigens, we propose that GS1 B4 and MPA should accompany each other.

J Bacteriol, 2002 Jul, 184(13), 3704 - 11
Functional interaction of region 4 of the extracytoplasmic function sigma factor FecI with the cytoplasmic portion of the FecR transmembrane protein of the Escherichia coli ferric citrate transport system; Mahren S et al.; Transcriptional regulation of the ferric citrate transport genes of Escherichia coli is initiated by the binding of ferric citrate to the outer membrane protein FecA . This binding elicits a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm, where the sigma factor FecI directs the RNA polymerase to the promoter upstream of the fecABCDE genes . An in vivo deletion analysis using a bacterial two-hybrid system assigned the interaction of the FecR and FecI proteins to the cytoplasmic portion of the FecR transmembrane protein and region 4 of FecI . Missense mutations randomly generated by PCR were localized to region 4 of FecI, and the mutants were impaired with regard to the interaction of FecR with FecI and fecB-lacZ transcription . The cloned region 4 of FecI interfered with fecB-lacZ transcription . Interaction of N-proximal regions of predicted FecR homologs with region 4 of predicted FecI homologs of Pseudomonas aeruginosa was demonstrated . The interaction was specific in that only cognate protein pairs interacted with each other; no interactions occurred between heterologous combinations of the P . aeruginosa proteins and between a P . aeruginosa FecI homolog and E . coli FecR . The results demonstrate that region 4 of FecI specifically binds FecR and that this binding is necessary for FecI to function as a sigma factor.

J Bacteriol, 2002 Jul, 184(13), 3614 - 22
Genetic variation at the O-antigen biosynthetic locus in Pseudomonas aeruginosa; Raymond CK et al.; The outer carbohydrate layer, or O antigen, of Pseudomonas aeruginosa varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described . Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus . We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains . DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region . Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis . The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome . The overall pattern underscores the plasticity of the P . aeruginosa genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences.

J Bacteriol, 2002 Jul, 184(13), 3605 - 13
Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa; Beatson SA et al.; Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa . We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr . Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production . We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasI::Tc) with a spontaneous deletion in vfr (S . A . Beatson, C . B . Whitchurch, A . B . T . Semmler, and J . S . Mattick, J . Bacteriol., 184:3598-3604, 2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket of Vfr . This allele (VfrDeltaEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility . Furthermore, structural analysis of Vfr and VfrDeltaEQERS in relation to E . coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrDeltaEQERS is only capable of responding to cAMP . We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

CLAO J, 2002 Apr, 28(2), 62 - 5
Bacterial keratitis and inflammatory corneal reactions: possible relations to contact lens oxygen transmissibility: the Harold A . Stein Lectureship 2001; Nilsson SE; PURPOSE: Conventional contact lenses, particularly when used for extended wear (EW), are still associated with a high incidence of microbial keratitis and inflammatory reactions . This article discusses the possible relationship of such complications to contact lens oxygen transmissibility (Dk/t) . METHODS: The literature, including our own work, is reviewed in regard to the binding of bacteria to the corneal epithelium and receptors likely to be associated with such binding, as well as the incidence of microbial keratitis and inflammatory reactions, in relation to conventional, lower Dk/t contact lenses and new, high Dk/t soft silicone hydrogel lenses . RESULTS: Receptors for lectins and, presumably, for bacteria such as Pseudomonas aeruginosa on the surface of the corneal epithelium are exposed in significantly higher numbers after wear of low Dk/t contact lenses than after wear of higher Dk/t lenses; and binding of P . aeruginosa follows the same pattern . Thus, it seems that high Dk/t lenses may reduce the risk of microbial keratitis . Published results of clinical trials of high Dk/t soft silicone hydrogel lenses worn for 7-day EW or 30-day continuous wear (CW), including our own 1-year study of 504 patients, are promising in so far as no cases of bacterial keratitis have yet been reported . In our study of high Dk/t silicone hydrogels, the incidence of inflammatory reactions, such as sterile infiltrates, was significantly lower than in studies of conventional soft lenses . Thus, it may be possible that inflammatory reactions also are related to oxygen transmissibility . CONCLUSIONS: High Dk/t contact lenses seem to reduce the rate of complications.

FEMS Immunol Med Microbiol, 2002 Jun 3, 33(2), 89 - 99
Enhancement of the protective efficacy of an oprF DNA vaccine against Pseudomonas aeruginosa; Price BM et al.; The outer membrane protein F gene (oprF) of Pseudomonas aeruginosa was recently shown by us to protect mice from P . aeruginosa chronic pulmonary infection when used as a DNA vaccine administered by three biolistic (gene gun) intradermal inoculations given at 2-week intervals . In the present study, we used two different strategies to improve the protective efficacy of the DNA vaccine . In the first strategy, mice were primed with two biolistic intradermal inoculations with the oprF vaccine and then were given a final intramuscular booster immunization containing either a synthetic peptide-keyhole limpet hemocyanin (KLH) conjugate or a chimeric influenza virus . Both the synthetic peptide conjugate and the chimeric virus contained peptide 10, a previously identified immunoprotective epitope of protein F . The second strategy involved the addition of a second outer membrane protein to the vaccine . DNA encoding a fusion protein comprised of the C-terminal half of protein F fused to OprI was administered by three biolistic intradermal inoculations . Challenge with P . aeruginosa in a chronic pulmonary infection model demonstrated that boosting with the chimeric virus (but not with peptide-KLH) or adding oprI to the DNA vaccine significantly enhanced protection as compared to that afforded by the oprF vaccine given alone . Thus, both strategies appear to augment the protection afforded by an oprF-only DNA vaccine.

JAMA, 2002 Jun 12, 287(22), 2958 - 67
Respiratory infections with Pseudomonas aeruginosa in children with cystic fibrosis: early detection by serology and assessment of risk factors; West SE et al.; CONTEXT: Patients with cystic fibrosis (CF) are susceptible to lower respiratory tract infections with Pseudomonas aeruginosa and typically acquire this organism in early childhood . Once P aeruginosa infection is established, eradication may be impossible, and progressive lung disease often aggravates morbidity and mortality risks . The ability to diagnose CF by genetic testing at birth makes it possible to determine the temporal sequence of events that result in P aeruginosa-associated pulmonary infections . OBJECTIVE: To evaluate the longitudinal relationship between the production of an antibody response against P aeruginosa and clinical factors associated with P aeruginosa pulmonary infections in patients with CF diagnosed in early life . DESIGN, SETTING, AND PATIENTS: Serum samples and oropharyngeal cultures (protocol cultures) were obtained at 6-month intervals from April 15, 1985, to April 15, 2000 (or for up to 180 months depending on their enrollment date) from 68 patients at 2 centers in Madison and Milwaukee, Wis, diagnosed through the Wisconsin CF Neonatal Screening Project, a longitudinal cohort study . Additional cultures were obtained at examining physicians' discretion (all cultures) . MAIN OUTCOME MEASURES: Time to serum IgG, IgA, and IgM antibody titer of at least 1:256 against P aeruginosa, assessed by enzyme-linked immunosorbent assay using cell lysate, exotoxin A, and elastase as antigens; time to organism isolation from respiratory samples; time to Wisconsin Cystic Fibrosis Radiograph (WCXR) score of 5 or more . RESULTS: The median time to an antibody titer of at least 1:256 was 17.8, 24.2, and 70.9 months for cell lysate, exotoxin A, and elastase, respectively . The rise of anti-cell lysate and anti-exotoxin A titers to 1:256 or more occurred a mean of 11.9 (P<.001) and 5.6 (P =.04) months, respectively, before the isolation of P aeruginosa for all cultures and 18.2 (P<.001) and 11.9 (P =.006) months, respectively, before protocol cultures . There was no significant difference between the rise of anti-cell lysate and anti-exotoxin A titer and a WCXR score of 5 or more (P =.24 and.32, respectively) . Treatment with long-term, non-Pseudomonas oral antibiotics and integration of CF infants with older, chronically infected patients were associated with a significantly increased risk of P aeruginosa pulmonary infection . CONCLUSIONS: In CF patients diagnosed through neonatal screening, P aeruginosa pulmonary infections occurred 6 to 12 months before the organism was isolated from respiratory secretions . The longitudinal monitoring of P aeruginosa antibody titers, in concert with WCXR score, should facilitate diagnosis and treatment of P aeruginosa pulmonary infections in young children with CF.

Mem Inst Oswaldo Cruz, 2002 Apr, 97(3), 387 - 94
Pseudomonas aeruginosa: study of antibiotic resistance and molecular typing in hospital infection cases in a neonatal intensive care unit from Rio de Janeiro City, Brazil; Loureiro MM et al.; This study had the objective of to analyze the demographic and bacteriologic data of 32 hospitalized newborns in an neonatal intensive care unit of a public maternity hospital in Rio de Janeiro city, Brazil, seized by Pseudomonas aeruginosa sepsis during a period ranged from July 1997 to July 1999, and to determine the antimicrobial resistance percentage, serotypes and pulsed field gel electrophoresis (PFGE) patterns of 32 strains isolated during this period . The study group presented mean age of 12.5 days, with higher prevalence of hospital infection in males (59.4%) and vaginal delivery (81.2%), than females (40.6%) and cesarean delivery (18.8%), respectively . In this group, 20 (62.5%) patients received antimicrobials before positive blood cultures presentation . A total of 87.5% of the patients were premature, 62.5% presented very low birth weight and 40.6% had asphyxia . We detected high antimicrobial resistance percentage to b-lactams, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline among the isolated strains . All isolated strains were classified as multi-drug resistant . Most strains presented serotype O11 while PFGE analysis revealed seven distinct clones with isolation predominance of a single clone (75%) isolated from July 1997 to June 1998.

Microbes Infect, 2002 May, 4(6), 613 - 20
Antibiotic resistance and virulence properties of Pseudomonas aeruginosa strains from mechanically ventilated patients with pneumonia in intensive care units: comparison with imipenem-resistant extra-respiratory tract isolates from uninfected patients; Di Martino P et al.; We investigated the epidemiology of antibiotic resistance and virulence properties among Pseudomonas aeruginosa clinical isolates collected in 1999 from patients hospitalized in the intensive care units of the centre hospitalier d'Orleans, in France . We compared the totality of the strains from mechanically ventilated patients with pneumonia (33 non-duplicate isolates, group 1) to 15 randomly chosen, imipenem-resistant, extra-respiratory tract isolates, collected from non-infected patients hospitalized in the same units (group 2) . The isolates were serotyped, typed by random amplified polymorphic DNA (RAPD), and screened for their pneumocyte cell adherence, cytotoxicity, and antibiotic resistance . A total of 35 RAPD profiles were found, and only two profiles were encountered in both groups, demonstrating a high genetic diversity . 84.8% of the group 1 and 93.3% of the group 2 isolates adhered to A549 cells . Three non-exclusive adhesive patterns were observed: a diffuse adhesion in 38 isolates, a localized adhesion in 14 isolates, and an aggregative adhesion in seven isolates . 78.8% of the group 1 and 93.3% of the group 2 isolates were cytotoxic . Considering all 48 isolates, there was a strong and statistically significant correlation between cytotoxicity and adherence . Among the three dominant serotypes, O:12 isolates were in majority avirulent, but the great majority of O:1 and all the O:11 isolates were found adherent and cytotoxic . Gentamicin was the least active antibiotic for both groups, and ceftazidime was the most active antibiotic for group 1 and amikacin for group 2 . The penicillinase production phenotype was significantly correlated with a decrease in P . aeruginosa virulence.

Clin Microbiol Infect, 2002 May, 8(5), 275 - 81
Selection of empiric therapy in patients with catheter-related infections; Rodriguez-Bano J; Catheter-related infections (CRI) are frequent and manifest in a wide range of clinical situations . A rational approach is necessary for the adequate management of these infections . Whenever a CRI is suspected, two main questions have to be addressed: whether to remove the catheter and whether to initiate empiric antimicrobial treatment . As the clinical diagnosis of CRI has a low specificity, the catheter should be removed only in circumstances such as severe or ongoing sepsis, persistent bacteremia, pulmonary or peripheral embolization, endocarditis, signs of tunnel infection, when the catheters or when the CRI is caused by fungi, Staphylococcus aureus or Pseudomonas aeruginosa are easy to replace among others . Exchanging the catheter through a guidewire is a frequent practice but is not recommended by some authors . Empiric antimicrobial treatment should be administered in any of the following situations: when the catheter is not removed, in the case of central venous or surgically implanted catheters and prosthetic implants, in the presence of severe sepsis, neutropenia or other immunodepressed status, suppurative phlebitis, embolization and acute endocarditis . Empriic antimicrobial treatment should include a glycopeptide (vancomycin or teicoplanin) as staphylococci are the most frequent cause of CRI . Adding an antipseudomonal agent, such as amikacin, aztreonam, ceftazidime, cefepime, piperacillin/tazobactam, or a carbapenem (depending on the local antimicrobial susceptibility data or antibiotic policy) is necessary in cases of neutropenia, burn patients, severe sepsis, or suspicion of contaminated infusate . Empiric treatment against Candida is not initially necessary in most cases . Empiric treatment should be replaced by specific therapy whenever possible.

Orbit, 1999 Jun, 18(2), 117 - 121
Orbital pachymengitis secondary to Pseudomonas aeruginosa; O'Halloran HS et al.; Pachymengitis is a very rare disorder that can present with multiple cranial neuropathies . The etiology can be inflammatory, infective or a combination of both, resulting in a thickening of the cranial dura and an obliteration of the individual layers of the meninges . We present a rare case of Pseudomonas aeruginosa pachymeningitis in the orbit that resulted in severe and permanent visual loss, in a patient after extensive sinus surgery .

Curr Eye Res, 2001 Dec, 23(6), 406 - 14
The role of IL-1beta in the regulation of IL-8 and IL-6 in human corneal epithelial cells during Pseudomonas aeruginosa colonization; Xue ML et al.; PURPOSE: Recent studies have shown that the levels of interleukin (IL)-1beta, IL-6 and IL-8 are associated with the severity of infectious diseases . The purpose of this study was to investigate whether IL-1beta regulates the expression of IL-6 and IL-8 in human corneal epithelial cells during Pseudomonas aeruginosa colonization . METHODS: Confluent immortalized human corneal epithelial cells were challenged with P . aeruginosa 6294 in the presence of anti-human IL-1beta antibody or matched control antibody . The cells were also challenged with recombinant IL-1beta protein without bacterial colonization . Expression of IL-1beta, IL-6 and IL-8 mRNA and protein was detected by reverse transcription (RT)-PCR and by enzyme-linked immunosorbent assay (ELISA), respectively . IL-1beta localization was determined by immunohistochemistry . RESULTS: Human corneal epithelial cells expressed low levels of IL-1beta and high levels of IL-6 and IL-8 during P . aeruginosa colonization . Addition of IL-1beta Ab resulted in a significant decrease (P < 0.05) in IL-8 protein expression at 4 h, 8 h and 12 h . Addition of IL-1beta Ab reduced IL-6 protein expression at 8 h and increased IL-6 protein expression at 12 h . Addition of recombinant IL-1beta protein alone strongly stimulated the expression of IL-8 and IL-6 . Immunohistochemical staining showed that IL-1beta protein was present both intracellularly and extracellularly in P . aeruginosa colonized cells . CONCLUSIONS: IL-1beta is able to modulate expression of both IL-6 and IL-8 at transcriptional and post-transcriptional levels in human corneal epithelial cells.

Life Sci, 2002 Jun 14, 71(4), 447 - 56
Differential effects of catecholamines on in vitro growth of pathogenic bacteria; Belay T et al.; Supplementation of minimal medium inoculated with bacterial cultures with norepinephrine, epinephrine, dopamine, or isoproterenol resulted in marked increases in growth compared to controls . Norepinephrine and dopamine had the greatest enhancing effects on growth of cultures of Pseudomonas aeruginosa and Klebsiella pneumoniae, while epinephrine and isoproterenol also enhanced growth to a lesser extent . The growth of Escherichia coli in the presence of norepinephrine was greater than growth in the presence of the three other neurochemicals used in the study . Growth of Staphylococcus aureus was also enhanced in the presence of norepinephrine, but not to the same degree as was the growth of gram negative bacteria . Addition of culture supernatants from E . coli cultures that had been grown in the presence of norepinephrine was able to enhance the growth of K . pneumoniae . Addition of the culture supernatant fluid culture from E . coli cultures that had been grown in the presence of norepinephrine did not enhance growth of P . aeruginosa or S . aureus . Culture supernatant fluids from bacteria other than E . coli grown in the presence of norepinephrine were not able to enhance the growth of any bacteria tested . The results suggest that catecholamines can enhance growth of pathogenic bacteria, which may contribute to development of pathogenesis; however, there is no uniform effect of catecholamines on bacterial growth.

FEMS Microbiol Lett, 2002 May 7, 210(2), 277 - 83
Pseudomonas aeruginosa galU is required for a complete lipopolysaccharide core and repairs a secondary mutation in a PA103 (serogroup O11) wbpM mutant; Dean CR et al.; Insertional inactivation of wbpM in Pseudomonas aeruginosa serogroup O11 strain PA103 resulted in mutants exhibiting three distinct lipopolysaccharide (LPS) phenotypes . One mutant, PA103 wbpM-C, had a truncated LPS core and lacked O antigen . These defects were not complemented by the cloned wbpM gene, suggesting a secondary mutation was present . When the wild-type galU gene was introduced into strain PA103 wbpM-C containing the cloned wbpM gene, both LPS defects were corrected . Construction of galU mutants in P . aeruginosa serogroups O11, O5, O6 and O17 strains led to truncation of the LPS core, indicating the involvement of GalU in P . aeruginosa LPS core synthesis.

J Endod, 2002 Apr, 28(4), 276 - 8
Bacteriologic evaluation of the effect of Nd:YAG laser irradiation in experimental infected root canals; Piccolomini R et al.; The aim of this study was to evaluate the efficacy of the Pumped Diodium-Nd:YAG laser in sterilizing contaminated root canals . After hand instrumentation, 30 teeth were inoculated with Actinomyces naeslundii CH-12 and 30 teeth with Pseudomonas aeruginosa ATCC 27853 and incubated for 24 h . The teeth were divided into three subgroups: subgroup A received no treatment; subgroup B was irradiated with laser (5 Hz for 15 s or 10 Hz for 15 s); and subgroup C was irrigated with 5.25% NaOCl . The number of viable bacteria in each group was evaluated by using the surface-spread plate technique . The results indicated an average of 34.0% decrease in colony-forming units for A . naeslundii CH-12 and 15.7% for P . aeruginosa ATCC 27853 with the 5 Hz/15 s laser treatment, and for the 10 Hz laser frequency, a decrease of the 77.4% for A . naeslundii CH-12 and 85.8% for P . aeruginosa ATCC 27853 . No bacteria were detected in the canals treated with 5.25% NaOCl . The results show an antibacterial effect of the Pumped Diodium Nd:YAG laser, depending on the radiation frequency . However, 5.25% NaOCl was more effective than either laser application.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Mar-Apr, (2), 81 - 3
{Opportunistic bacteria detected in cultivated mussels}; Beleneva IA et al.; As many as 8 Listeria monocytogenes strains, 12 Pseudomonas aeruginosa strains and 5 Staphylococcus aureus strains were isolated from mussels Mytilus edulis, grown on special installations in the Trinity Bay of the Gulf of Peter the Great, the Sea of Japan . The isolated cultures proved to be highly resistant to a number of antibiotics . Many strains displayed DNAase and haemolytic activity . The cultures of L . monocytogenes, S . aureus and P . aeruginosa also had high lipase, protease and lecithinase activity . The organism of the mussels seems to be a confinement for these bacteria under study.

Nat Med, 2002 Jun, 8(6), 588 - 93
CD1d-dependent macrophage-mediated clearance of Pseudomonas aeruginosa from lung; Nieuwenhuis EE et al.; CD1d-restricted T cells are implicated as key players in host defense against various microbial infections . However, the mechanisms involved and the role they play, if any, at the mucosal surfaces where pathogenic infections are initiated is unknown . In a murine pneumonia model established by intranasal application of Pseudomonas aeruginosa, CD1d(-/-) mice showed markedly reduced pulmonary eradication of P . aeruginosa compared with wild-type mice; this was associated with significantly lower amounts of macrophage inflammatory protein-2 and reduced numbers of neutrophils within the bronchoalveolar lavage fluid . Corollarily, treatment of mice with alpha-galactosylceramide--a lipid that activates CD1d-restricted T cells--increased the amount of interferon-gamma; this was associated with rapid pulmonary clearance through enhanced phagocytosis of P . aeruginosa by alveolar macrophages . These results reveal a crucial role played by CD1d-restricted T cells in regulating the antimicrobial immune functions of macrophages at the lung mucosal surface.

J Chemother, 1991 Jan, 3 Suppl 1, 208 - 12
Imipenem/cilastatin as empirical treatment of severe infections in compromised patients; Biglino A et al.; Imipenem/cilastatin was administered as empirical treatment to 22 patients with severe infections, at dosages of 2 or 4 g/day, either alone or in combination with an aminoglycoside . The majority of patients had underlying diseases causing various degrees of immune system dysfunction . The overall cure rate was 77.2%, without significant differences according to the type of pathogen or dosage schedule; however one of the two observed failures was due to a resistant Pseudomonas aeruginosa . Strains recovered from improved patients were all sensitive to the drug . Only mild adverse effects were evidenced, without any alteration of laboratory parameters . Imipenem/cilastatin may be useful as monotherapy in empirical treatment of severe infections in compromised patients.

Environ Sci Technol, 2002 May 15, 36(10), 2171 - 7
Cometabolism of cis-1,2-dichloroethene by aerobic cultures grown on vinyl chloride as the primary substrate; Verce MF et al.; An aerobic enrichment culture was grown on vinyl chloride (VC) as the sole source of carbon and energy . In the absence of VC, the enrichment culture cometabolized cis-1,2-dichloroethene (cDCE) and, to a lesser extent, trans1,2-dichloroethene (tDCE), beginning with oxidation to the corresponding DCE-epoxides . When provided with VC (1.3 mM) and cDCE (0.2-0.3 mM), the enrichment culture cometabolized repeated additions of cDCE for over 85 days . Cometabolism of repeated additions of tDCE was also demonstrated but at a lower ratio of nongrowth substrate to VC . VC-grown Pseudomonas aeruginosa MF1 (previously isolated from the enrichment culture) also readily cometabolizes cDCE, with an observed transformation capacity (Tc,obs) of 0.82 micromol of cDCE/mg of total suspended solids (TSS) . When provided with VC and cDCE, MF1 did not begin cometabolizing cDCE until nearly all of the VC was consumed . The presence of cDCE reduces the maximum specific rate of VC utilization . A kinetic model was developed that describes these phenomena via Monod parameters for substrate and nongrowth substrate, plus inactivation and inhibition coefficients . MF1 did not show any cometabolic activity on tDCE or trichloroethene and very limited activity on 1,1-DCE (Tc,obs = 2 x 10(-5) micromol/mg TSS) . Above 40 microM, tDCE and TCE noticeably increased the maximum specific rate of VC utilization, even though neither compound was consumed during or after VC consumption . High concentrations of 1,1-DCE (950 microM) completely inhibited VC biodegradation . As there is currently no evidence for aerobic biodegradation of cDCE as a sole source of carbon and energy, the results of this study provide a potential explanation for in situ disappearance of cDCE when the only other significant substrate available is VC . It is fortuitous that the VC-grown cultures tested exhibit their highest cometabolic activity toward cDCE, because it is the predominant DCE isomer formed during anaerobic reductive dechlorination of trichloroethene and tetrachloroethene.

Acta Crystallogr D Biol Crystallogr, 2002 Jun, 58(Pt 6 Pt 2), 1045 - 7 Epub 2002 May 29.
Towards structural understanding of feedback control of arginine biosynthesis: cloning and expression of the gene for the arginine-inhibited N-acetyl-L-glutamate kinase from Pseudomonas aeruginosa, purification and crystallization of the recombinant enzyme and preliminary X-ray studies; Fernandez-Murga ML et al.; N-Acetyl-L-glutamate kinase (NAGK) catalyzes the second step in the pathway of arginine biosynthesis in microorganisms and plants . In many species, it is the pathway-controlling enzyme and is subject to feedback inhibition by arginine . The gene for the best characterized arginine-inhibitable NAGK, that from Pseudomonas aeruginosa, has been cloned in a pET22 plasmid and overexpressed in Escherichia coli . The enzyme was purified in three steps to 95% purity and was shown by cross-linking to form dimers . It was crystallized by the hanging-drop vapour-diffusion method at 277 K in the presence of ADP, Mg and N-acetyl-L-glutamate . The crystallization solution contained 0.1 M sodium cacodylate pH 6.5, 150-170 mM magnesium acetate and 13% polyethylene glycol 8000 . Prismatic crystals of maximum dimension approximately 0.5 mm diffract to 2.75 A resolution and belong to space group P1 (unit-cell parameters a = 71.86, b = 98.78, c = 162.9 A, alpha = 91.49, beta = 92.03, gamma = 107.56 degrees ) . Packing density considerations agree with 6-18 NAGK monomers in the asymmetric unit, with a corresponding solvent content of 79-36% . Self-rotation function calculations confirm the space group and suggest the presence of 3-7 dimers in the unit cell.

Thorax, 2002 Jun, 57(6), 559 - 60
Spread of an epidemic Pseudomonas aeruginosa strain from a patient with cystic fibrosis (CF) to non-CF relatives; McCallum SJ et al.; Colonisation with Pseudomonas aeruginosa is common in adults with cystic fibrosis (CF) and there is increasing evidence that transmissible strains may cross colonise patients . However, transmission of these strains by social contact to healthy non-CF individuals has not been described . A case is presented where an adult CF patient colonised by an epidemic P aeruginosa strain infected her parents with subsequent morbidity.

Gene, 2002 May 1, 289(1-2), 131 - 9
Synonymous codon usage in Pseudomonas aeruginosa PA01; Grocock RJ et al.; Pseudomonas aeruginosa PA01 has a large (6.7 Mbp) genome with a high (67%) G+C content . Codon usage in this species is dominated by this compositional bias, with the average G+C content at synonymously variable third positions of codons being 83% . Nevertheless, there is some variation of synonymous codon usage among genes . The nature and causes of this variation were investigated using multivariate statistical analyses . Three trends were identified . The major source of variation was attributable to genes with unusually low G+C content that are probably due to horizontal transfer . A lesser trend among genes was associated with the preferential use of putatively translationally optimal codons in genes expressed at high levels . In addition, genes on the leading strand of replication were on average more G+T-rich . Our findings contradict the results of two previous analyses, and the reasons for the discrepancies are discussed.

Microb Ecol, 2001 Jul, 42(1), 99 - 107
Impact of Heavy Metals on the Selective Phenotypical Markers of Pseudomonas aeruginosa; Hassen A et al.; The aim of this study was to characterize the impact of heavy metals on phenotypical markers of Pseudomonas aeruginosa . Twenty-two isolates of P . aeruginosa, either clinical (20) or secondary treated wastewater (2), were used to inoculate micro-ecosystems of sterile distilled water or secondary waste effluent in the presence of subminimal inhibitory concentrations of a variety of heavy metals commonly encountered in the aquatic naturally habitat (Ca2+, Co2+, Cr3+, Cu2+, Hg2+, Ni2+, Zn2+) . Micro-ecosystems were exposed to visible light at laboratory temperature and individual strains were reisolated after a 1-, 3-, or 6-month period . The re-isolates (129) were characterized using hierarchical classification analysis in order to define affinities among variants of P . aeruginosa . Subsequently, discriminant analysis was used to detect eventual relationships among the different phenotypical markers studied . Results of the hierarchical classification, based on qualitative or quantitative approaches, showed clearly that incubation of P . aeruginosa in the presence of heavy metals altered the studied phenotypical markers, namely serotype, phage type, MIC of metals, and pyocin type . Discriminant analysis showed that the studied phenotypical markers could be classified into four clusters: C1 (L1 and L2 phage types, Hg tolerance and/or resistance, S2 serotype), C2 (P2 pyocin type, Cd tolerance and/or resistance, S1 serotype), C3 (Co and Cr tolerance and/or resistance) and C4 (P1 pyocin type, Ni, Zn, and Cu tolerance and/or resistance).

J Biol Chem, 2002 Aug 9, 277(32), 29253 - 9 Epub 2002 May 28.
Crystal structure of the MexR repressor of the mexRAB-oprM multidrug efflux operon of Pseudomonas aeruginosa; Lim D et al.; MexR is a member of the MarR family of bacterial transcriptional regulators and is the repressor for the MexAB-OprM operon, which encodes a tripartite multidrug efflux system in Pseudomonas aeruginosa . Mutations in MexR result in increased resistance to multiple antibiotics due to overexpression of this efflux system . We have determined the crystal structure of MexR to 2.1-A resolution in the absence of effector . The four copies of the MexR dimer in the asymmetric unit are observed in multiple conformations . Analysis of these conformational states in the context of a model of the MexR-DNA complex proposed in this study suggests that an effector-induced conformational change may inhibit DNA binding by reducing the spacing of the DNA binding domains . The inhibited conformation is exhibited by one of the four MexR dimers, which contains an ordered C-terminal tail from a neighboring monomer inserted between its DNA binding domains and which we propose may resemble the MexR-effector complex . Our results indicate that MexR may differ from the other described member of this family, MarR, in the nature of its effector, mode of DNA binding, and mechanism of regulation.

Am J Respir Cell Mol Biol, 2002 Jun, 26(6), 731 - 8
Subinhibitory bismuth-thiols reduce virulence of Pseudomonas aeruginosa; Wu CL et al.; Pseudomonas aeruginosa is a common pathogen in mechanically ventilated patients and produces a wide array of virulence factors . Bismuth-thiols (BTs) are active in vitro against all bacterial lung pathogens, including P . aeruginosa . The objective of these studies was to examine the biochemical and morphologic effects of sublethal BT concentrations on P . aeruginosa and to evaluate virulence in cell culture . Bismuth-dimercaprol, at a fraction of the minimal inhibitory concentration, reduced alginate expression by 67% in P . aeruginosa, whereas subinhibitory bismuth-ethanedithiol (BisEDT) reduced alginate by 92% in P . syringae . BisEDT effects on lipopolysaccharide content and type III secreted cytoxins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis . Subinhibitory BisEDT reduced cell-associated lipopolysaccharide, and inhibited processing of the secreted cytotoxic protein ExoU . BisEDT-induced outer membrane blebbing and aggregation of cytoplasmic material was noted in electron microscopy . Virulence of P . aeruginosa was assessed by adherence to epithelial cells and sensitivity to serum killing . BisEDT inhibited adherence of P . aeruginosa to 16HBE14o- cells by 28% and to a collagen matrix by 53% . BisEDT-treated bacteria were also 100-fold more sensitive to serum bactericidal activity . In summary, low BT concentrations affect P . aeruginosa in a variety of ways, the combination of which may help prevent or resolve respiratory tract infection.

Clin Infect Dis, 2002 Jun 15, 34(12), 1558 - 63 Epub 2002 May 23.
Control-group selection importance in studies of antimicrobial resistance: examples applied to Pseudomonas aeruginosa, Enterococci, and Escherichia coli; Harris AD et al.; We aimed to illustrate the importance of control-group selection on the results of risk factor analysis for (1) imipenem-resistant Pseudomonas aeruginosa, (2) vancomycin-resistant enterococci (VRE), and (3) ampicillin-sulbactam-resistant Escherichia coli . Case patients were compared with 2 different control groups: patients with the susceptible form of the organism (type 1), and control patients among whom the case patients arose during the same period as the case patients (type 2) . Comparison of case patients who had imipenem-resistant P . aeruginosa with type-1 control patients identified use of imipenem (odds ratio {OR}, 27.1) and quinolones (OR, 3.25) as a risk factor for selection of antimicrobial resistance, and comparison of the same case patients with type-2 control patients identified imipenem (OR, 6.34) . When case patients with VRE were compared with type-1 and with type-2 control patients, use of vancomycin was identified as a risk factor (OR, 4.38 and 2.77, respectively) . Comparison of case patients who had ampicillin-sulbactam-resistant E . coli compared with type-1 control patients identified ampicillin-sulbactam (OR, 2.71) and quinolones (OR, 2.72), and comparison with type-2 control patients identified ampicillin-sulbactam (OR, 1.68) . The selection of control patients from the potentially suboptimal control type 1 can falsely identify certain antibiotics and overestimate the OR of the resistance-defining antibiotic.

J Bacteriol, 2002 Jun, 184(12), 3377 - 84
Characterization and regulation of the gbuA gene, encoding guanidinobutyrase in the arginine dehydrogenase pathway of Pseudomonas aeruginosa PAO1; Nakada Y et al.; The arginine dehydrogenase (or oxidase) pathway catabolically converts arginine to succinate via 2-ketoglutarate and 4-guanidinobutyrate (4-GB) with the concomitant formation of CO(2) and urea . Guanidinobutyrase (GBase; EC 3.5.3.7) catalyzes the conversion of 4-guanidinobutyrate to 4-aminobutyrate and urea in this pathway . We investigated the structure and regulation of the gene for GBase (designated gbuA) of Pseudomonas aeruginosa PAO1 and characterized the gbuA product . The gbuA and the adjacent gbuR genes were cloned by functional complementation of a gbuA9005 mutant of strain PAO1 defective in 4-GB utilization . The deduced amino acid sequence of GbuA (319 amino acids; M(r) 34,695) assigned GBase to the arginase/agmatinase family of C-N hydrolases . Purified GbuA was a homotetramer of 140 kDa that catalyzed the specific hydrolysis of 4-GB with K(m) and K(cat) values of 49 mM and 1,012 s(-1,) respectively . The divergent gbuR gene, which shared the intergenic promoter region of 206 bp with gbuA, encoded a putative regulatory protein (297 amino acids; M(r) 33,385) homologous to the LysR family of proteins . Insertional inactivation of gbuR by a gentamicin resistance cassette caused a defect in 4-GB utilization . GBase and gbuA'::'lacZ fusion assays demonstrated that this gbuR mutation abolishes the inducible expression of gbuA by exogenous 4-GB, indicating that GbuR participates in the regulation of this gene . Northern blotting located an inducible promoter for gbuA in the intergenic region, and primer extension localized the transcription start site of this promoter at 40 bp upstream from the initiation codon of gbuA . The gbuRA genes at the genomic map position of 1547000 are unlinked to the 2-ketoarginine utilization gene kauB at 5983000, indicative of at least two separate genetic units involved in the arginine dehydrogenase pathway.

J Bacteriol, 2002 Jun, 184(12), 3268 - 75
FpvA receptor involvement in pyoverdine biosynthesis in Pseudomonas aeruginosa; Shen J et al.; Alignment of the Pseudomonas aeruginosa ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca . 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of P . putida . Deletion of fpvA from the chromosome of P . aeruginosa reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g., pvdD) associated with the biosynthesis of the pyoverdine peptide moiety . Wild-type fpvA restored pvd expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of fpvA encoding a receptor lacking the N terminus of the mature protein did not . The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium . These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in P . aeruginosa.

Med Hypotheses, 2002 Apr, 58(4), 327 - 31
Treatment of post-burns bacterial infections by bacteriophages, specifically ubiquitous Pseudomonas spp . notoriously resistant to antibiotics; Ahmad SI; Post-burn microbial infections are a major problem in recovering from the trauma of third-degree burns, and the survival of patients can depend upon the severity of the burn and the infections encountered . Within 24 hours, patients can start suffering from opportunistic bacterial attacks, which can vary from simple infection, such as those easily treatable by antibiotics, to more complicated types, which may have natural or acquired resistance to drugs . Infection by multiple drug-resistant bacteria can create additional complexity to the problem . As an alternative to treating bacterial infections by antibiotics, bacteriophages have been in use in certain parts of the world, such as at Tbilisi in Georgia and in Poland, and this approach has now been more widely recognized . Results have shown that phage therapy has an 80% success rate against Enterococcus infections and up to 90% against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae . Here it is proposed that bacteriophages can effectively be used for the treatment of post-burn infections, particularly the ubiquitous opportunistic pathogens, Pseudomonas spp., known to be notoriously resistant to a variety of antibiotics . This kind of treatment may be of particular importance in Third World countries where the incidence of burns and infections, due to lack of stringent safety regulations and proper hygiene respectively, may be more common and where cocktails of antibiotics may be less affordable . Phages that can possibly be employed in the treatment and their advantages compared to the use of antibiotics are also highlighted .

Biochem Soc Trans, 2002 Apr, 30(2), 111 - 5
Antimicrobial activity of antiproteinases; Sallenave JM; Low-molecular-mass neutrophil elastase inhibitors have been shown to be important in the control of lung inflammation . In addition to inhibiting the enzyme neutrophil elastase, these low-molecular-mass compounds (10 kDa) have been shown to have other activities . For example, secretory leucocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor/SKALP (skin-derived antileucoproteinase)/elafin have also been shown to have "defensin"-like antimicrobial activities . Indeed, these inhibitors have antimicrobial properties in vitro against bacteria, fungi and, potentially, HIV . In addition, we have shown, using an adenovirus-mediated gene transfer overexpression strategy, that elafin is also active against Pseudomonas aeruginosa infection in mice in vivo . The mechanism of action is currently under investigation . In addition to these direct or indirect effects on microbes, it has been shown that lipopolysaccharide is able to up-regulate SPLI production in macrophages in vitro, and that the addition of recombinant SLPI to human monocytes or the transfection of macrophages with SPLI can down-regulate pro-inflammatory mediators such as tumour necrosis factor, presumably to limit self-damaging excessive inflammation . Using viral gene transfer vectors, we are currently investigating the potential of these inhibitors in various models of inflammation in vivo.

J Immunol, 2002 Jun 1, 168(11), 5756 - 63
IL-18 contributes to host resistance against infection with Pseudomonas aeruginosa through induction of IFN-gamma production; Huang X et al.; Pseudomonas aeruginosa keratitis destroys the cornea in susceptible (B6), but not resistant (BALB/c) mice . To determine mechanisms mediating resistance, the role of IFN-gamma, IL-12, and IL-18 was tested in BALB/c mice . RT-PCR analysis detected IFN-gamma mRNA expression levels in cornea that were significantly increased at 1-7 days postinfection . IL-18 mRNA was detected constitutively in cornea and, at 1-7 days postinfection, levels were elevated significantly, while no IL-12 mRNA was similarly detected . To test whether IL-18 contributed to IFN-gamma production, mice were treated with anti-IL-18 mAb . Treatment decreased corneal IFN-gamma mRNA levels, and bacterial load and disease increased/worsened, compared with IgG-treated mice . To stringently examine the role of IFN-gamma in bacterial killing, knockout (-/-) vs wild-type (wt) mice also were tested . All corneas perforated, and bacterial load was increased significantly in -/- vs wt mice . Because disease severity was increased in IFN-gamma(-/-) vs IL-18-neutralized mice, and since IL-18 also induces production of TNF, we tested for TNF-alpha in both groups . ELISA analysis demonstrated significantly elevated corneal TNF-alpha protein levels in IFN-gamma(-/-) vs wt mice after infection . In contrast, RT-PCR analysis of IL-18-neutralized vs IgG-treated infected mice revealed decreased corneal TNF-alpha mRNA expression . Next, to resolve whether TNF was required for bacterial killing, TNF-alpha was neutralized in BALB/c mice . No difference in corneal bacterial load was detected in neutralized vs IgG-treated mice . These data provide evidence that IL-18 contributes to the resistance response by induction of IFN-gamma and that IFN-gamma is required for bacterial killing.

FEMS Microbiol Lett, 2002 Apr 23, 210(1), 73 - 80
Identification of in vivo essential genes from Pseudomonas aeruginosa by PCR-based signature-tagged mutagenesis; Lehoux DE et al.; We adapted PCR-based signature-tagged mutagenesis (STM) to Pseudomonas aeruginosa . A collection of 1056 mutants was screened in a chronic lung infection rat model . Thirteen mutants were confirmed to be attenuated . Analysis revealed that these STM mutants represented transposon insertions into eight genes previously described in databases, three genes encoding proteins sharing identity with hypothetical proteins and two genes that shared no significant identity with sequences in databases . Five strains mutated in genes involved in protein degradation, stress tolerance, cation transport, ABC transporter, and an unknown protein were shown to be highly attenuated when tested individually in the rat chronic lung infection model.

Chang Gung Med J, 2002 Mar, 25(3), 144 - 52
Amniotic membrane transplantation for pseudomonal keratitis with impending perforation; Chen JH et al.; BACKGROUND: To determine whether amniotic membrane transplantation (AMT) can be used as adjunctive therapy to promote wound healing and prevent perforation in bacterial keratitis caused by Pseudomonas aeruginosa . METHODS: We report on 6 eyes from 6 patients with bacterial keratitis caused by Pseudomonas aeruginosa associated with prominent stromal melting and extensive stromal loss . AMT was performed after treatment with fortified antibiotics for at least 1 week . The mean follow-up period was 12.8+/-2.5 months . RESULTS: The lesion became sterile in all but 1 case for which AMT was performed . Rapid reepithelialization and decreased inflammation was observed in 5 cases, with complete reepithelialization occurred at 9.4+/-2.1 days postoperatively . The amniotic membrane dissolved in the remaining case with active, extensive corneal infection and persistent epithelial defect; this case finally received evisceration due to intractable glaucoma . In all other cases, after AMT treatment, lesions did not extend, stromal loss was limited, and considerable stromal thickness was preserved . CONCLUSION: AMT may be considered an alternative method for treating pseudomonal keratitis, especially when stromal melting and loss are extensive, and the infection has been controlled.

Acta Paediatr, 2002, 91(3), 303 - 6
Septicaemia due to glucose non-fermenting, gram-negative bacilli other than Pseudomonas aeruginosa in children; Ladhani S et al.; Bloodstream infections due to non-fermenting Gram-negative bacilli other than Pseudomonas aeruginosa (NF-GNB) are uncommon in children but their incidence is reported to be increasing . The aim of this study was to determine the characteristics of such infections in children in a London teaching hospital . All paediatric patients with positive NF-GNB blood cultures and clinical evidence of sepsis between July 1995 and June 2000 were included in the study . A total of 10278 blood cultures was performed, of which 356 (3.5%) represented clinically significant episodes of bacteraemia . Of these, 12 (0.1%) were due to NF-GNB . Nine of the 12 (75%) patients were receiving haemodialysis for end-stage renal failure (ESRF) . Only one patient was receiving immunosuppressive therapy and none was neutropenic or had any malignancy . An intravascular catheter was identified as the focus of infection in all 12 cases . Stenotrophomonas maltophilia was the most common organism isolated (67%) . Six patients were successfully treated with antibiotics alone . Four others received antibiotics, but also required line removal, and two patients responded to line removal without the need for antibiotics . CONCLUSION: An association was found between ESRF and NF-GNB infections, possibly related to the requirement for long-term catheters for dialysis . Antibiotic treatment alone was only successful in half the cases of catheter-related NF-GNB septicaemia, while removal of the infected catheter ensured complete cure in the cases where antibiotic treatment alone did not suffice.

Am J Otolaryngol, 2002 May-Jun, 23(3), 142 - 7
The use of otic powder in the treatment of acute external otitis; Goldenberg D et al.; BACKGROUND: Acute external otitis (AEO) is a painful condition that results as a secondary infection of macerated skin and subcutaneous tissues of the external auditory canal . The most commonly causative microorganisms are Pseudomonas aeruginosa and Staphylococcus aureus . Classic management strategies include moisture prevention, cleansing of the canal and administration of topical antimicrobial agents in drop form, such as aminoglycosides and quinolones, at times in combination with steroid solutions . The objective of this study was to evaluate and compare the efficacy of topical otic powder, tobramycin drops and ciprofloxacin drops in patients suffering from AEO . MATERIALS AND MEASURES: A randomized prospective trial was performed to determine the efficacy of Auricularum powder (dexamethasone 10 mg, oxytetracycline HCl 90,000 U, polymyxin B Sulfate 100,000 U, nystatin 1,000,000 U; Trima, Serolam Laboratories, Germany) compared with ciprofloxacin (Ciloxan, Alcon Laboratories, Fort Worth, TX) and tobramycin (Tobrex, Alcon Laboratories) drops for the treatment of AEO . One hundred twenty patients who presented with signs and symptoms of AEO were enrolled . Inclusion criteria were: AEO diagnosed by an otolaryngologist, patient age 18 years, no prior treatment with other drops or systemic antibiotics, no sensitivity to any of the drugs used or their contents, and no perforation of the tympanic membrane . All patients were instructed to avoid moisture and wetness of the ear during the course of their treatment . After we received informed consent, a swab culture was taken, and the patient was randomly assigned topical treatment for 14 days . RESULTS: Eighty-six percent of those treated with Auricularum powder were cured at day 3-4 after initial treatment . Seventy-seven percent of those treated with ciprofloxacin drops, and fifty-six percent of those treated with tobramycin were cured at that time . All 120 patients were cured by day 14 . CONCLUSION: The results show that topical treatment with Auricularum powder is an effective and rapid method for the treatment of AEO . Ciloxan also was effective in the treatment of AEO and relieved symptoms quickly and efficiently in a short period of time . Tobrex was effective in treating AEO, but our results show that relief of symptoms was slower than with the other drugs .

Antimicrob Agents Chemother, 2002 Jun, 46(6), 2035 - 7
Nonradiolabeling assay for WaaP, an essential sugar kinase involved in biosynthesis of core lipopolysaccharide of Pseudomonas aeruginosa; Zhao X et al.; waaP is present in the lipopolysaccharide (LPS) core gene clusters of a wide range of gram-negative bacteria, and is an essential gene in Pseudomonas aeruginosa . The WaaP protein is a sugar kinase that adds phosphate to heptose I in the core oligosaccharide . This study describes the standardization and utility of a chemiluminescence-based enzyme-linked immunosorbent assay for the detection of WaaP kinase activity . Important features of the assay include high sensitivity, the preparation of dephosphorylated LPS as a substrate, and the use of monoclonal antibody 7-4 that specifically recognizes phosphate substituents in the LPS core.

Antimicrob Agents Chemother, 2002 Jun, 46(6), 2032 - 4
Efficacy of beta-lactams for treating experimentally induced pneumonia due to a carbapenem-hydrolyzing metallo-beta-lactamase-producing strain of Pseudomonas aeruginosa; Bellais S et al.; A rat pneumonia model was established with a Pseudomonas aeruginosa strain that produced the plasmid-encoded metallocarbapenemase VIM-2 . A significant decrease in lung bacterial titers was observed when imipenem, cefepime, ceftazidime, and piperacillin-tazobactam were given at the highest doses recommended for humans, despite their high MICs . Aztreonam at high doses produced a similar decrease in bacterial titers.

Antimicrob Agents Chemother, 2002 Jun, 46(6), 1940 - 5
Broad-spectrum bactericidal activity of Ag(2)O-doped bioactive glass; Bellantone M et al.; Bioactive glass has found extensive application as an orthopedic and dental graft material and most recently also as a tissue engineering scaffold . Here we report an initial investigation of the in vitro antibacterial properties of AgBG, a novel bioactive glass composition doped with Ag(2)O . The bacteriostatic and bactericidal properties of this new material and of two other bioactive glass compositions, 45S5 Bioglass and BG, have been studied by using Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus as test microorganisms . Concentrations of AgBG in the range of 0.05 to 0.20 mg of AgBG per ml of culture medium were found to inhibit the growth of these bacteria . Not only was AgBG bacteriostatic, but it also elicited a rapid bactericidal action . A complete bactericidal effect was elicited within the first hours of incubation at AgBG concentrations of 10 mg ml(-1) . 45S5 Bioglass and BG had no effect on bacterial growth or viability . The antibacterial action of AgBG is attributed exclusively to the leaching of Ag(+) ions from the glass matrix . Analytical measurements rule out any contribution to AgBG-mediated bacterial killing by changes in pH or ionic strength or the dissolution of other ionic species from the biomaterials . Our observations of the dissolution profiles of Ag(+) from AgBG in the presence and absence of bacteria are consistent with silver accumulation by the bacteria.

Antimicrob Agents Chemother, 2002 Jun, 46(6), 1880 - 6
Identification of a series of tricyclic natural products as potent broad-spectrum inhibitors of metallo-beta-lactamases; Payne DJ et al.; This work describes the discovery and characterization of a novel series of tricyclic natural product-derived metallo-beta-lactamase inhibitors . Natural product screening of the Bacillus cereus II enzyme identified an extract from a strain of Chaetomium funicola with inhibitory activity against metallo-beta-lactamases . SB236050, SB238569, and SB236049 were successfully extracted and purified from this extract . The most active of these compounds was SB238569, which possessed K(i) values of 79, 17, and 3.4 microM for the Bacillus cereus II, Pseudomonas aeruginosa IMP-1, and Bacteroides fragilis CfiA metallo-beta-lactamases, respectively, yet none of the compounds exhibited any inhibitory activity against the Stenotrophomonas maltophilia L-1 metallo-beta-lactamase (50% inhibitory concentration > 1,000 microM) . The lack of activity against angiotensin-converting enzyme and serine beta-lactamases demonstrated the selective nature of these compounds . The crystal structure of SB236050 complexed in the active site of CfiA has been obtained to a resolution of 2.5 A . SB236050 exhibits key polar interactions with Lys184, Asn193, and His162 and a stacking interaction with the indole ring of Trp49 in the flap, which is in the closed conformation over the active site groove . SB236050 and SB238569 also demonstrate good antibacterial synergy with meropenem . Eight micrograms of SB236050 per ml gave rise to an eightfold drop in the MIC of meropenem for two clinical isolates of B . fragilis producing CfiA, making these strains sensitive to meropenem (MIC < or = 4 microg/ml) . Consequently, this series of metallo-beta-lactamase inhibitors exhibit the most promising antibacterial synergy activity so far observed against organisms producing metallo-beta-lactamases.

Antimicrob Agents Chemother, 2002 Jun, 46(6), 1837 - 44
Activity of novispirin G10 against Pseudomonas aeruginosa in vitro and in infected burns; Steinstraesser L et al.; The emergence of multidrug-resistant microbes has serious implications for managing infection and sepsis and has stimulated efforts to develop alternative treatments, such as antimicrobial peptides . The objective of this study was to test a designer peptide, novispirin G10, against multidrug-resistant microorganisms . By two-stage radial diffusion assays, its activity against such organisms compared favorably with that of standard antibiotics and other antimicrobial peptides . It killed bacteria very rapidly, was nonhemolytic, and was relatively noncytotoxic . The peptide induced an immediate, massive efflux of potassium from Pseudomonas aeruginosa, suggesting that it altered the permeability of its inner membrane . The presence of human serum reduced but did not eliminate its activity . We tested the in vivo activity of novispirin G10 in rats with an infected, partial-thickness burn that covered 20% of their total body surface area . The burned area was seeded with 10(6) CFU of a Silvadene-resistant P . aeruginosa strain, and 24 h later a single treatment with 0, 1, 3, or 6 mg of synthetic novispirin G10 (n = 16 at each concentration) per kg was given intradermally . Significant bacterial killing (P < 0.0001) was evident within 4 h in each peptide group compared to controls receiving vehicle . Antimicrobial peptides such as novispirin G10 may provide a useful alternative or adjunct to standard antibiotic agents in treating burns or other wound infections.

Indian J Exp Biol, 2001 Dec, 39(12), 1318 - 21
Effect of metal ions on growth of Pseudomonas aeruginosa and siderophore and protein production; Gupta CP et al.; Pseudomonas aeruginosa (GRC1) isolated from potato rhizosphere, grew better on succinate medium than tryptic soy medium and produced hydroxamate type of siderophore in iron-deficient succinate medium . When the strain GRC1 was grown in the presence of different metal ion compounds, viz . ZnSO4, MnSO4, MnCl2 and FeCl3 at 6 and 12 microM concentrations individually, ZnSO4 (12 microM) promoted siderophore production but suppressed the growth and protein content of test organism . MnCl2 and FeCl3 (12 microM) enhanced the growth, whereas MnCl2 and MnSO4 (12 microM) induced protein contents of strain GRC1.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 1025 - 35
Evaluation of different carbon and nitrogen sources in production of rhamnolipids by a strain of Pseudomonas aeruginosa; Santos AS et al.; Culture conditions involving variations in carbon and nitrogen sources and different C:N ratios were examined with the aim of increasing productivity in the process of rhamnolipid synthesis by Pseudomonas aeruginosa . In addition to the differences in productivity, the use of different carbon sources resulted in several proportions related to the types of rhamnolipids synthesized (monorhamnolipids and dirhamnolipids) . Furthermore, the variation in nutrients, mainly the nitrogen source, resulted in different amounts of virulence factors, as phenazines and extracellular proteins . The data point out a new concern in the choice of substrate to be used for rhamnolipid production by P . aeruginosa: toxic byproducts.

J Chemother, 2002 Apr, 14(2), 175 - 80
Pharmacokinetic parameters of ciprofloxacin (500 mg/5 mL) oral suspension in critically ill patients with severe bacterial pneumonia: a comparison of two dosages; Debon R et al.; The authors determined the pharmacokinetic parameters of a new immediate-release ciprofloxacin suspension in tube-fed intensive care patients with bacterial pneumonia, to compare two dosage regimens: 500 mg b.i.d and 750 mg b.i.d . in this prospective clinical trial . The 20 patients were critically ill and on mechanical ventilation and enteral feeding with bacterial pneumonia . They were randomized to receive two different ciprofloxacin dosages: 500 mg b.i.d (group 1) versus 750 mg b.i.d . (group 2) . Blood samples were collected from these patients after reaching steady-state and the pharmacokinetic parameters were determined . The mean (range) serum steady-state concentration at 2 h after enteral administration was: C(max 500) = 2.6 (1.2-4.3) mg/L in group 1 and C(max 750) = 3.5 (1.5-5.9) mg/L in group 2 . The mean (range) calculated 12-h area under the serum concentration was high in both groups: AUC(0-12 (500)) = 24.7 (12.9-36.2) mg.h/L in group 1 and AUC(0-12 (750)) = 28.9 (18.3-47.5) mg.h/L in group 2 . In conclusion, ciprofloxacin oral suspension was well absorbed via nasogastric route in intensive care patients with severe pneumonia, achieving reliable pharmacokinetic parameters for most of the pathogens and important cost reduction compared to intravenous delivery . However, with less susceptible pathogens such as Staphylococcus aureus or Pseudomonas aeruginosa, higher dosages than 750 mg b.i.d . should be given.

Mem Inst Oswaldo Cruz, 2002 Mar, 97(2), 205 - 7
Occurrence of cryptosporidial oocysts and giardia cysts in bottled mineral water commercialized in the city of Campinas, State of São Paulo, Brazil; Franco RM et al.; The consumption of bottled mineral water has significantly increased in Brazil so that it is in the interest of public health to determine the parasitological and microbiological status of some brands of Brazilian mineral water available in the town of Campinas, Sao Paulo, Brazil . For this purpose, detection of protozoa by direct immunofluorescence technique and microbiological parameters were determined for each specimen after membrane filtration . Giardia cysts were not present while cryptosporidial oocysts were detected in two samples . The counts of protozoa varied from 0.2 to 0.5 oocysts/l . The detected level of Pseudomonas aeruginosa and heterotrophic bacteria reflected the level of organic enrichment of the water.

New Microbiol, 2002 Apr, 25(2), 223 - 9
Elastase and alkaline protease production by Pseudomonas aeruginosa strains: comparison of two procedures; Yagci A et al.; Pseudomonas aeruginosa is an opportunistic pathogen that can cause fatal infections in immunocompromised hosts . The virulence of P . aeruginosa is associated with the presence of various extracellular factors like elastase and alkaline protease . These enzymes are suggested to contribute to tissue destruction and assist bacterial invasion during infection . Therefore it seems likely that determination of these virulence factors will be an important prognostic marker in the near future especially for follow up of cystic fibrosis patients, to start antimicrobial agents that are directly or indirectly inhibit microbial growth or virulence factor production . Herein, we suggest a simple test procedure to be used in routine laboratories for estimation of elastase and alkaline protease levels and compare them with quantitative methods in the literature . We detected the amount of elastase and alkaline protease in 49 clinical P . aeruginosa isolates by comparing agar plate method and colorimetric assay . The resulting values were in the range reported in the literature and differed from one strain to another(elastase: 0-1390 mg/ml, alkaline protease: 0- 770 mg/ml) . Linear relationships were found when assays compared in pairs and significant correlation coefficients were obtained(r>0.788 for alkaline protease, p<0.0001- r>0.926 for elastase, p<0.0001) . Our method can be applied in laboratories regardless of the availability of technical equipment.

Pharmacotherapy, 2002 May, 22(5), 569 - 77
Pharmacokinetic and pharmacodynamic evaluation of two dosing regimens for piperacillin-tazobactam; Kim MK et al.; STUDY OBJECTIVE: To compare the pharmacokinetic and pharmacodynamic profiles of two dosing regimens for piperacillin-tazobactam against commonly encountered pathogens . The regimens compared were piperacillin 4.0 g-tazobactam 0.5 g administered every 8 hours, and piperacillin 3.0 g-tazobactam 0.375 g administered every 6 hours . DESIGN: Multiple-dose, open-label, randomized, crossover study . SETTING: Clinical research center at Hartford Hospital . SUBJECTS: Twelve healthy volunteers . INTERVENTION: The two dosing regimens for piperacillin-tazobactam were administered intravenously in crossover design . Blood was sampled after the third dose . MEASUREMENTS AND MAIN RESULTS: Drug concentrations were determined by a validated high-performance liquid chromatography assay . The percentage of time above minimum inhibitory concentration (%T>MIC) for piperacillin was calculated for a range of MIC values . The maximum concentration (Cmax), area under the concentration-time curve (AUC0-tau), and total clearance of piperacillin differed significantly between the two study regimens, as did the Cmax, AUC0-tau, volume of distribution, and total clearance of tazobactam (p<0.05) . The piperacillin 4.0 g-tazobactam 0.5 g regimen provided 40-50% T>MIC for MIC values 8-16 microg/ml; a similar value for the piperacillin 3.0 g-tazobactam 0.375 g regimen was 16-32 microg/ml . CONCLUSION: Although statistically significant differences in the pharmacodynamic profile were noted for the regimens, both provide adequate T>MIC against commonly encountered pathogens considered susceptible to piperacillin-tazobactam . However, for treatment of Pseudomonas aeruginosa infection, combination therapy or higher-dosage regimens (e.g., piperacillin 3.0 g-tazobactam 0.375 g every 4 hours, piperacillin 4.0 g-tazobactam 0.5 g every 6 hours, or continuous-infusion piperacillin 12 g-tazobactam 1.5 g/day) may be a prudent option when full MIC data are unavailable.

J Perinat Med, 2002, 30(2), 149 - 57
Neonatal sepsis of nosocomial origin: an epidemiological study from the "Grupo de Hospitales Castrillo"; Lopez Sastre JB et al.; A prospective multicenter study was designed to assess the frequency, etiology, and mortality of nosocomial neonatal sepsis diagnosed between 1996 and 1997 in the neonatology services of 27 acute-care hospitals in Spain ("Grupo de Hospitales Castrillo") . Nosocomial sepsis is defined in the literature using chronological criteria (> 3-7 days of life at the onset of symptoms); accordingly, there is the possibility of including late-onset maternally acquired sepsis or of excluding early-onset nosocomial sepsis (< 3-7 days of life) . For these reasons, in this study, cases of nosocomial sepsis that developed at < or = 3-7 days after birth (early onset) were also recorded and maternally acquired sepsis diagnosed beyond 3-7 days of life were excluded . Using these criteria in a total of 30,993 admissions to the neonatal units of the participating hospitals, the nosocomial sepsis rate was 2.1% with an incidence density of 0.89 per 1000 patient days . Sepsis rate was significantly more frequent among very low birth weight (VLBW) infants (15.6%) than among those weighing > or = 1500 g (1.16%) (P < 0.001) . Fifty-eight percent of all isolates were Gram-positive organisms, mainly Staphylococcus epidermidis (42%) . Gram-negative organisms were isolated in 29.5% of cases (Escherichia coli and Klebsiella spp . were the most commonly isolated pathogens) and fungal infections in 12%, with absolute predominance of Candida spp . The overall mortality rate was 11.8% and the following subgroups had significantly higher (P < 0.001) mortality rates: sepsis caused by Gram-negative organisms (19% vs . 5.1% in Gram-positive pathogens) and sepsis caused by Pseudomonas aeruginosa (33.3% vs . 9.4% for the total number of sepsis caused by the remaining causative pathogens) . Sepsis caused by S . epidermidis showed a significantly lower mortality rate (5.5%) compared with overall sepsis for the remaining etiologies (14.2%) (P < 0.001) . In VLBW infants, the mortality rate was significantly higher than in infants weighing > 1500 g (17.3% vs . 6.5%, P < 0.001).

Chemotherapy, 2002 May, 48(2), 59 - 63
Comparative pharmacokinetic and pharmacodynamic profile of piperacillin/tazobactam 3.375G Q4H and 4.5G Q6H; Mattoes HM et al.; When piperacillin/tazobactam has been used to treat hospitalized patients with serious infections, including nosocomial pneumonia caused by Pseudomonas aeruginosa, it has usually been dosed at 3.375 g q4h to provide serum concentrations above commonly encountered organisms' MICs (T > MIC) for at least 40-50% of the dosing interval . Pharmacodynamic principles suggest that a similar efficacy can be realized with extended dosing intervals when a larger dose (e.g . 4.5 g q6h) is administered, which was the objective of this study . Twelve healthy volunteers, 29.4 +/- 8.9 years of age, were enrolled in this multiple-dose, open-labeled, randomized, two-period crossover study . Blood samples were collected after the third dose and concentrations of piperacillin/tazobactam were determined with a validated HPLC method . Pharmacokinetic profiles were determined by noncompartment analysis . T > MIC of piperacillin was calculated for a range of MIC values . Piperacillin/tazobactam was well tolerated in 11 subjects who completed both regimens . The C(max), T(1/2), K, and AUC of P were significantly different according to a paired t test (p < 0.05) between two study regimens . Significant differences (p < 0.05) in tazobactam regimens were noted for C(max), and AUC . The piperacillin/tazobactam regimen of 4.5 g q6h achieved a 44% T > MIC for MIC values of < or = 16 microg/ml, while the 3.375-gram q4h regimen achieved 42% T > MIC, for MIC values of < or = 32 microg/ml . Dosage regimens for treating serious infections can be extended safely and effectively to 4.5 g q6h and obtain at least 40-50% T > MIC in the coverage of pathogens implicated with serious infections, including P . aeruginosa .

Infect Immun, 2002 Jun, 70(6), 3004 - 11
Identification of a quorum-sensing signal molecule in the facultative intracellular pathogen Brucella melitensis; Taminiau B et al.; Brucella melitensis is a gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans . This facultative intracellular pathogen invades and survives within both professional and nonprofessional phagocytes . A dichloromethane extract of spent culture supernatant from B . melitensis induces bioluminescence in an Escherichia coli acyl-homoserine lactone (acyl-HSL) biosensor strain based upon the activity of the LasR protein of Pseudomonas aeruginosa . HPLC fractionation of the extract, followed by mass spectrometry, identified the major active molecule as N-dodecanoylhomoserine lactone (C12-HSL) . This is the first report of the production of an acyl-HSL by an intracellular pathogen . The addition of synthetic C12-HSL to an early log phase culture of either B . melitensis or Brucella suis 1330 reduces the transcription of the virB operon, which contains virulence genes known to be required for intracellular survival . This mimics events seen during the stationary phase of growth and suggests that quorum sensing may play a role in the control of virulence in Brucella.

Infect Immun, 2002 Jun, 70(6), 2837 - 45
Identification of the Pseudomonas aeruginosa 1244 pilin glycosylation site; Comer JE et al.; Previous work (P . Castric, F . J . Cassels, and R . W . Carlson, J . Biol . Chem . 276:26479-26485, 2001) has shown the Pseudomonas aeruginosa 1244 pilin glycan to be covalently bound to a serine residue . N-terminal sequencing of pilin fragments produced from endopeptidase treatment and identified by reaction with a glycan-specific monoclonal antibody indicated that the glycan was present between residue 75 and the pilin carboxy terminus . Further sequencing of these peptides revealed that serine residues 75, 81, 84, 105, 106, and 108 were not modified . Conversion of serine 148, but not serine 118, to alanine by site-directed mutagenesis, resulted in loss of the ability to carry out pilin glycosylation when tested in an in vivo system . These results showed the pilin glycan to be attached to residue 148, the carboxy-terminal amino acid . The carboxy-proximal portion of the pilin disulfide loop, which is adjacent to the pilin glycan, was found to be a major linear B-cell epitope, as determined by peptide epitope mapping analysis . Immunization of mice with pure pili produced antibodies that recognized the pilin glycan . These sera also reacted with P . aeruginosa 1244 lipopolysaccharide as measured by Western blotting and enzyme-linked immunosorbent assay.

Clin Experiment Ophthalmol, 2002 Jun, 30(3), 196 - 9
Gene expression of pro-inflammatory cytokines and chemokines in mouse eye infected with Pseudomonas aeruginosa; Xue ML et al.; Ocular infection with Pseudomonas aeruginosa triggers extensive host inflammatory response and corneal damage . The purpose of present study was to investigate the gene expression of pro-inflammatory mediators interleukin (IL)-1 beta, IL-6, tumour necrosis factor-alpha (TNF-alpha),macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (KC) in the mouse eye challenged with P . aeruginosa . Scratched mouse corneas were infected with three phenotypes of P . aeruginosa individually.Total RNA was extracted from mouse eyes at 4 h, 8 h,16 h and 24 h post-challenge . Single stranded cDNA was synthesized from total RNA by reverse transcription and then subjected to polymerase chain reaction (PCR) using specific primers for IL-1 beta, IL-6, TNF-alpha, MIP-2 and KC . Results revealed three patterns of cytokines and chemokines expression in response to ocular infection with three phenotypes of P . aeruginosa . Ocular infection with the invasive strain induced the highest levels of IL-1 beta, IL-6, MIP-2 and KC mRNA, followed by the infection with the cytotoxic strain.Ocular infection with the CLARE strain induced the lowest levels of IL-1 beta, IL-6, MIP-2 and KC mRNA . The expression of TNF-alpha mRNA was very low and irregular following P . aeruginosa challenge.These data indicate that over-expression of pro-inflammatory cytokines and chemokines may represent a vigorous immune response and therefore may contribute to corneal damage during P . aeruginosa infection.

Int J Antimicrob Agents, 2002 May, 19(5), 377 - 82
Investigation into the selection frequency of resistant mutants and the bacterial kill rate by levofloxacin and ciprofloxacin in non-mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients; Gillespie T et al.; The frequency by which resistant Pseudomonas aeruginosa strains could be selected was compared for two antibiotics, levofloxacin and ciprofloxacin . Seven distinct strains were cultured on plates containing 1x, 2x, 4x and 8x the minimum inhibitory concentration (MIC) of the antibiotic under investigation . Resistant mutants were more readily isolated by growth on culture plates that contained ciprofloxacin, and the resulting MIC of the resistant mutant was also more frequently increased . Time-kill studies on comparable strains where the MIC for both antibiotics had increased by at least fourfold showed no difference between the two agents.

Biochim Biophys Acta, 2002 Apr 29, 1596(2), 336 - 45
Crystal structure of the double azurin mutant Cys3Ser/Ser100Pro from Pseudomonas aeruginosa at 1.8 A resolution: its folding-unfolding energy and unfolding kinetics; Okvist M et al.; Azurin is a cupredoxin, which functions as an electron carrier . Its fold is dominated by a beta-sheet structure . In the present study, azurin serves as a model system to investigate the importance of a conserved disulphide bond for protein stability and folding/unfolding . For this purpose, we have examined two azurin mutants, the single mutant Cys3Ser, which disrupts azurin's conserved disulphide bond, and the double mutant Cys3Ser/Ser100Pro, which contains an additional mutation at a site distant from the conserved disulphide . The crystal structure of the azurin double mutant has been determined to 1.8 A resolution(2), with a crystallographic R-factor of 17.5% (R(free)=20.8%) . A comparison with the wild-type structure reveals that structural differences are limited to the sites of the mutations . Also, the rates of folding and unfolding as determined by CD and fluorescence spectroscopy are almost unchanged . The main difference to wild-type azurin is a destabilisation by approximately 20 kJ x mol(-1), constituting half the total folding energy of the wild-type protein . Thus, the disulphide bond constitutes a vital component in giving azurin its stable fold.

Cloning Stem Cells, 2002, 4(1), 91 - 102
Production of human monoclonal and polyclonal antibodies in TransChromo animals; Ishida I et al.; We have developed TransChromo (TC) technology, which enables the introduction of megabase-sized segments of DNA into cells . We have used this approach to derive mice that carry megabases of human DNA by the use of a human chromosome fragment (HCF) as a vector . TC technology has been applied to the construction of the TC Mouse,trade mark which incorporates entire human immunoglobulin (hIg) loci . TC Mouse expresses a fully diverse repertoire of hIgs, including all the subclasses of IgGs (IgG1-G4) . Immunization of the TC Mouse with various human antigens produced antibody responses comprised of human antibodies . Furthermore, it was possible to obtain hybridoma clones expressing fully human antibodies specific for the target human antigen . However, because of the instability of the Igkappa locus-bearing HCF2, the efficiency of hybridoma production was less than one-tenth of that observed in normal mice . An instant solution to this problem was to cross-breed the Kirin TC Mouse carrying the HCF14, which was stable in mouse cells, with the Medarex YAC-transgenic mouse carrying about 50% of the hIgVkappa gene segments as a region that is stably integrated into the mouse genome . The resulting mouse, dubbed the KM Mouse, performed as well as normal mice with regard to immune responsiveness and efficiency of hybridoma production . Another application of TC technology is the production of polyclonal antibodies in large animals such as chickens and cows . To test the efficacy of human polyclonal antibodies derived from TC animals, feasibility studies were performed using antisera and purified gamma-globulin from TC mice immunized with Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), or Japanese encephalitis virus (JEV) . The TC mouse-derived antisera and gamma-globulin showed a much higher titer and efficacy in terms of the neutralizing activity of the pathogens in vitro and in vivo than either human serum or gamma-globulin prepared from human blood.

J Antimicrob Chemother, 2002 May, 49(5), 863 - 6
Antibiotic tolerance of peritoneal bacterial isolates in dialysis fluids; Zelenitsky S et al.; The objective was to test antibiotic activity against Staphylococcus epidermidis and Pseudomonas aeruginosa in fresh dianeal fluid and spent dialysate from patients undergoing chronic ambulatory peritoneal dialysis . MICs and MBCs were measured and compared with those in standard broth . For S . epidermidis, there was some reduction in cefazolin and vancomycin activity in dialysis fluids . For P . aeruginosa, ciprofloxacin and tobramycin MICs increased significantly, and all isolates were tolerant to ceftazidime and piperacillin in dialysis fluids . Dialysis fluids can significantly impair antibiotic activity . The clinical implications warrant further study of antibiotic pharmacodynamics in the treatment of peritonitis.

J Antimicrob Chemother, 2002 May, 49(5), 745 - 55
Clarithromycin suppresses lipopolysaccharide-induced interleukin-8 production by human monocytes through AP-1 and NF-kappa B transcription factors; Kikuchi T et al.; Erythromycin and other macrolides are effective for the treatment of chronic inflammatory airway diseases such as diffuse panbronchiolitis (DPB) and chronic sinusitis . The effect of macrolides in DPB is suggested to be anti-inflammatory rather than antibacterial . We investigated the effects of clarithromycin on interleukin-8 (IL-8) production using human peripheral monocytes and the human monocytic leukaemia cell line, THP-1 . Bacterial extracts from Escherichia coli, Pseudomonas aeruginosa and Helicobacter pylori, as well as E . coli-derived lipopolysaccharide (LPS), induced IL-8 production . Clarithromycin suppressed this production in a dose-dependent manner in both monocytes and THP-1 cells (49.3-75.0% inhibition at 10 mg/L) . A luciferase reporter gene assay with plasmids containing a serially deleted IL-8 promoter fragment showed that both the activator protein-1 (AP-1) and/or the nuclear factor-kappa B (NF-kapp aB) binding sequences were responsible for the LPS and clarithromycin responsiveness of the IL-8 promoter . Consistently, in an electromobility shift assay, LPS increased the specific binding of both AP-1 and NF-kappaB, whereas clarithromycin suppressed it . Moreover, LPS and clarithromycin regulated three other promoters that have either the NF-kappa B or the AP-1 binding sequences: two synthetic (pAP-1-Luc and pNF-kappa B-Luc) and one naturally occurring (ELAM-Luc) . Our results indicate that clarithromycin modified inflammation by sup-pressing IL-8 production and that clarithromycin may affect the expression of other genes through AP-1 and NF-kappa B . In addition to treatment of airway diseases, the anti-inflammatory effect of macrolides may be beneficial for the treatment of other inflammatory diseases such as chronic gastritis caused by H . pylori.

J Bacteriol, 2002 Jun, 184(11), 3027 - 33
Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system; Cosson P et al.; Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors . P . aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains . In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P . aeruginosa strains . The virulent wild-type strain PAO1 was shown to inhibit growth of D . discoideum . Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells . Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role . Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D . discoideum cells . By using this simple model system, we predicted that certain antibiotic-resistant mutants of P . aeruginosa should show reduced virulence . This result was confirmed in a rat model of acute pneumonia . Thus, D . discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P . aeruginosa.

J Bacteriol, 2002 Jun, 184(11), 3000 - 7
Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa; Franklin MJ et al.; Alginate is an extracellular polysaccharide produced by mucoid strains of Pseudomonas aeruginosa that are typically isolated from the pulmonary tracts of chronically infected cystic fibrosis patients . Alginate is a linear polymer of D-mannuronate and L-guluronate with O-acetyl ester linkages on the O-2 and/or O-3 position of the mannuronate residues . The presence of O-acetyl groups plays an important role in the ability of the polymer to act as a virulence factor, and the algF, algJ, and algI genes are known to be essential for the addition of O-acetyl groups to alginate . To better understand the mechanism of O acetylation of alginate, the cellular locations of the AlgI, AlgJ, and AlgF proteins were determined . For these studies, defined nonpolar algI, algJ, and algF deletion mutants of P . aeruginosa strain FRD1 were constructed, and each mutant produced alginate lacking O-acetyl groups . Expression of algI, algJ, or algF in trans in the corresponding mutant complemented each O acetylation defect . Random phoA (alkaline phosphatase {AP} gene) fusions to algF, algJ, and algI were constructed . All in-frame fusions to algF and algJ had AP activity, indicating that both AlgF and AlgJ were exported to the periplasm . Immunoblot analysis of spheroplasts and periplasmic fractions showed that AlgF was released with the periplasmic contents but that AlgJ remained with the spheroplast fraction . An N-terminal sequence analysis of AlgJ showed that its putative AlgJ signal peptide was not cleaved, suggesting that AlgJ is anchored to the cytoplasmic membrane by its uncleaved signal peptide . AP gene fusions were also used to map the membrane topology of AlgI, and the results suggest that it is an integral membrane protein with seven transmembrane domains . These results suggest that AlgI-AlgJ-AlgF may form a complex in the membrane that is the reaction center for O acetylation of alginate.

Microb Drug Resist, 2002 Spring, 8(1), 9 - 13
Biochemical characterization of a novel extended-spectrum beta-lactamase from Pseudomonas aeruginosa 802; Rejiba S et al.; Pseudomonas aeruginosa 802 was isolated at Rabta hospital in Tunis and was resistant to extended-spectrum cephalosporins and aztreonam . It produced a pI 7.6 extended-spectrum beta-lactamase (ESBL) . The ESBL, named LBT 802, was purified to homogeneity by filtration on Sephadex G-75 followed by CM-Sepharose chromatography and high-performance liquid chromatography (HPLC) on a TSK-gel SP-5PW column . The LBT 802 enzyme had a molecular mass of 30 kDa . It showed a broad-substrate profile by hydrolyzing benzylpenicillin, ampicillin, cephalothin, cephaloridine, cefotaxime, ceftriaxone, and cefpirome but not ceftazidime, cefoxitin, imipenem, or aztreonam . The highest hydrolytic efficiency (Vmax/Km) was obtained for ampicillin, cephalothin, cephaloridine, and benzylpenicillin . Among extended-spectrum cephalosporins the best substrate was ceftriaxone followed by cefotaxime and cefpirome . LBT 802 activity was inhibited by clavulanic acid, sulbactam, imipenem, cefoxitin, and aztreonam . It showed its lowest Ki values for clavulanic acid, imipenem and sulbactam.

Curr Microbiol, 2002 Jun, 44(6), 425 - 30
Tight regulation and modulation via a C1-regulated promoter in Escherichia coli and Pseudomonas aeruginosa; Schofield DA et al.; We describe the development and analysis of a novel promoter system regulated by the bacteriophage P1 temperature-sensitive C1 repressor . Using transcriptional fusions to the lacZ reporter gene to monitor gene expression, we show that the ratio of induction/repression can be up to 1500-fold in Escherichia coli . The promoters exhibited extremely tight repression and could be modulated over a range of temperatures . The utility of the promoter system was tested in Pseudomonas aeruginosa . C1 effectively repressed transcription; however, only modest induction was achieved . To increase the levels of induction, the amount of c1 was modulated at the mRNA level by using a LacI-regulated promoter . This resulted in a 59-fold induction in gene expression under inducing conditions . As the promoter system was constructed in a broad-host range vector and utilized the C1 repressor from a broad-host range phage, the system will provide the potential for controlled gene expression in Gram-negative bacteria.

J Biol Chem, 2002 Jul 19, 277(29), 25947 - 56 Epub 2002 May 08.
Accumulation of the lipid A precursor UDP-2,3-diacylglucosamine in an Escherichia coli mutant lacking the lpxH gene; Babinski KJ et al.; The lpxH gene encodes the UDP-2,3-diacylglucosamine-specific pyrophosphatase that catalyzes the fourth step of lipid A biosynthesis in Escherichia coli . To confirm the function of lpxH, we constructed KB21/pKJB5 . This strain contains a kanamycin insertion element in the chromosomal copy of lpxH, complemented by plasmid pKJB5, which is temperature-sensitive for replication and harbors lpxH(+) . KB21/pKJB5 grows at 30 degrees C but loses viability at 44 degrees C, demonstrating that lpxH is essential . CDP-diglyceride hydrolase (Cdh) catalyzes the same reaction as LpxH in vitro but is non-essential and cannot compensate for the absence of LpxH . The presence of Cdh in cell extracts interferes with the LpxH assay . We therefore constructed KB25/pKJB5, which contains both an in-frame deletion of cdh and a kanamycin insertion mutation in lpxH, covered by pKJB5 . When KB25/pKJB5 cells are grown at 44 degrees C, viability is lost, and all in vitro LpxH activity is eliminated . A lipid migrating with synthetic UDP-2,3-diacylglucosamine accumulates in KB25/pKJB5 following loss of the covering plasmid at 44 degrees C . This material was converted to the expected products, 2,3-diacylglucosamine 1-phosphate and UMP, by LpxH . Pseudomonas aeruginosa contains two proteins with sequence similarity to E . coli LpxH . The more homologous protein catalyzes UDP-2,3-diacylglucosamine hydrolysis in vitro . The corresponding gene complements KB25/pKJB5 at 44 degrees C, but the less homologous gene does not . The accumulation of UDP-2,3-diacylglucosamine in our lpxH mutant is consistent with the observation that the lipid A disaccharide synthase LpxB, the next enzyme in the pathway, cannot condense two UDP-2,3-diacylglucosamine molecules, but instead utilizes UDP-2,3-diacylglucosamine as its donor and 2,3-diacylglucosamine 1-phosphate as its acceptor.

J Appl Microbiol, 2002, 92 Suppl, 55S - 64S
Mechanisms of bacterial biocide and antibiotic resistance; Poole K; Resistance to antibiotics is increasingly commonplace amongst important human pathogens . Although the mechanism(s) of resistance vary from agent to agent they typically involve one or more of: alteration of the drug target in the bacterial cell, enzymatic modification or destruction of the drug itself, or limitation of drug accumulation as a result of drug exclusion or active drug efflux . While most of these are agent specific, providing resistance to a single antimicrobial or class of antimicrobial, there are currently numerous examples of efflux systems that accommodate and, thus, provide resistance to a broad range of structurally unrelated antimicrobials -- so-called multidrug efflux systems . Resistance to biocides is less common and likely reflects the multiplicity of targets within the cell as well as the general lack of known detoxifying enzymes . Resistance typically results from cellular changes that impact on biocide accumulation, including cell envelope changes that limit uptake, or expression of efflux mechanisms . Still, target site mutations leading to biocide resistance, though rare, are known . Intriguingly, many multidrug efflux systems also accommodate biocides (e.g . triclosan) such that strains expressing these are both antibiotic- and biocide-resistant . Indeed, concern has been expressed regarding the potential for agents such as triclosan to select for strains resistant to multiple clinically-relevant antibiotics . Some of the better characterized examples of such multidrug efflux systems can be found in the opportunistic pathogen Pseudomonas aeruginosa where they play an important role in the noted intrinsic and acquired resistance of this organism to antibiotics and triclosan . These tripartite pumps include an integral inner membrane drug-proton antiporter, an outer membrane- and periplasm-spanning channel-forming protein and a periplasmic link protein that joins these two . Expression of efflux genes is governed minimally by the product of a linked regulatory gene that is in most cases the target for mutation in multidrug resistant strains hyperexpressing these efflux systems . Issues for consideration include the natural function of these efflux systems and the therapeutic potential of targeting these systems in combating acquired multidrug resistance.

Rofo, 2002 May, 174(5), 588 - 92
{Bronchial artery embolization for therapy of pulmonary bleeding in patients with cystic fibrosis}; Thalhammer A et al.; INTRODUCTION: Acute pulmonary emergencies in patient with cystic fibrosis (CF) can be found in cases of pneumothorax as well as hemoptysis . If the bleeding cannot be stopped by conservative methods, an embolization of the bronchial arteries should be done . MATERIALS AND METHOD: 11 patients were embolized using a combination of PVA particles and microcoils . RESULTS: From January 1996 to June 2001 17 bronchial arteries in 11 patients were embolized . 7 patients suffered from chronical hemoptysis, 4 patients had an acute hemoptysis . In 4 patients both sides were embolized, in 3 patients only one side . The remaining 4 patients needed a second intervention, embolizing the other side . The primary embolizated bronchial artery was still closed in all 4 patients . In 1 patient the selective catheterization of a bronchial artery was not successful, thus the embolization could not be carried out . 1 patient died 5 days after the intervention due to a fulminant pneumonia (Pseudomonas aeruginosa) without recurrent bleeding . In two patients atypical branches from intercostal arteries feeding the bronchial arteries were detected and successfully embolized . All patients profited from the therapy, as bleeding could be stopped or at least be reduced . 3 patients suffered from back pain during or after intervention . There were no severe complications like neurological deficiencies or necroses . CONCLUSION: The embolization of bronchial arteries using particles and microcoils is an effective intervention with a low complication rate in pulmonary bleeding of CF patients.

Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6907 - 12 Epub 2002 May 07.
CFTR is a pattern recognition molecule that extracts Pseudomonas aeruginosa LPS from the outer membrane into epithelial cells and activates NF-kappa B translocation; Schroeder TH et al.; Immune cells are activated during cellular responses to antigen by two described mechanisms: (i) direct uptake of antigen and (ii) extraction and internalization of membrane components from antigen-presenting cells . Although endocytosis of microbial antigens by pattern recognition molecules (PRM) also activates innate immunity, it is not known whether this involves extraction and internalization of microbial surface components . Epithelial cells on mucosal surfaces use a variety of receptors that are distinct from the classical endocytic PRM to bind and internalize intact microorganisms . Nonclassical receptor molecules theoretically could act as a type of endocytic PRM if these molecules could recognize, bind, extract, and internalize a pathogen-associated molecule and initiate cell signaling . We report here that the interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the outer core oligosaccharide of the lipopolysaccharide (LPS) in the outer membrane of Pseudomonas aeruginosa satisfies all of these conditions . P . aeruginosa LPS was specifically recognized and bound by CFTR, extracted from the organism's surface, and endocytosed by epithelial cells, leading to a rapid (5- to 15-min) and dynamic translocation of nuclear transcription factor NF-kappa B . Inhibition of epithelial cell internalization of P . aeruginosa LPS prevented NF-kappa B activation . Cellular activation depended on expression of wild-type CFTR, because both cultured Delta F508 CFTR human airway epithelial cells and lung epithelial cells of transgenic-CF mice failed to endocytose LPS and translocate NF-kappa B . CFTR serves as a critical endocytic PRM in the lung epithelium, coordinating the effective innate immune response to P . aeruginosa infection.

Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 7072 - 7 Epub 2002 May 07.
Siderophore-mediated signaling regulates virulence factor production in Pseudomonasaeruginosa; Lamont IL et al.; Numerous bacteria secrete low molecular weight compounds termed siderophores that have a high affinity for iron ions . Siderophores have a well-documented role as iron-scavenging chemicals, chelating iron ions in the environment whereupon the ferrisiderophores reenter the bacterial cells by means of specific cell-surface receptors . The iron is then released for incorporation into bacterial proteins . Here we show that in addition to its role as an iron-scavenger, the siderophore pyoverdine that is secreted by Pseudomonas aeruginosa regulates the production of at least three virulence factors (exotoxin A, an endoprotease, and pyoverdine itself), which are major contributors to the ability of this bacterium to cause disease . Regulation occurs through a transmembrane signaling system that includes an outer membrane receptor for ferripyoverdine, a signal-transducing protein that is predicted to extend from the periplasm into the cytoplasm, and a sigma factor . Expression of genes that form part of the regulon is triggered by pyoverdine so that this siderophore acts as a signaling molecule to control the production of secreted products . Recognition that a siderophore acts as a signaling molecule has important implications for the understanding of interactions between bacterial cells.

Chemosphere, 2002 Apr, 47(3), 333 - 42
Fixed-bed study for lanthanide (La, Eu, Yb) ions removal from aqueous solutions by immobilized Pseudomonas aeruginosa: experimental data and modelization; Texier AC et al.; A fixed-bed study was carried out by using cells of Pseudomonas aeruginosa immobilized in polyacrylamide gel as a biosorbent for the removal of lanthanide (La, Eu, Yb) ions from aqueous solutions . The effects of superficial liquid velocity based on empty column, particle size, influent concentration and bed depth on the lanthanum breakthrough curves were investigated . Immobilized biomass effectively removed lanthanum from a 6 mM solution with a maximum adsorption capacity of 342 micromolg(-1) (+/-10%) corresponding closely to that observed in earlier batch studies with free bacterial cells . The Bohart and Adams sorption model was employed to determine characteristic parameters useful for process design . Results indicated that the immobilized cells of P . aeruginosa enable removal of lanthanum, europium and ytterbium ions from aqueous effluents with significant and similar maximum adsorption capacities . Experiments with a mixed cation solution showed that the sequence of preferential biosorption was Eu3+ > or = Yb3+ > La3+ . Around 96+/-4% of the bound lanthanum was desorbed from the column and concentrated by eluting with a 0.1 M EDTA solution . The feasibility of regenerating and reusing the biomass through three adsorption/desorption cycles was suggested . Neural networks were used to model breakthrough curves performed in the dynamic process . The ability of this statistical tool to predict the breakthrough times was discussed.

Biotechnol Bioeng, 2002 Jun 20, 78(6), 699 - 707
Simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa; Hori K et al.; The feasibility of the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids, as a novel approach to reduce their production costs, was demonstrated by the cultivation of Pseudomonas aeruginosa IFO3924 . Fairly large amounts of PHAs and rhamnolipids were obtained from the bacterial cells and the culture supernatant, respectively . Decanoate was a more suitable carbon source than ethanol and glucose for the simultaneous production, although glucose was suitable for cell growth without an induction period under pH control . The kind of carbon source affected PHA monomer composition markedly and PHA molecular weight slightly . Monorhamnolipids and dirhamnolipids were included in the rhamnolipids extracted from the culture supernatant using decanoate, glucose, or ethanol as the carbon source . Both PHAs and rhamnolipids were synthesized after the growth phase . PHA content in the cell reached a maximum when the carbon source was exhausted . After exhaustion of the carbon source, PHA content decreased rapidly, but rhamnolipid synthesis, which followed PHA synthesis, continued . This resulted in a time lag for the attainment of maximum levels of PHAs and rhamnolipids . The reusability of the cells used in rhamnolipid production was evaluated in the repeated batch culture of P . aeruginosa IFO3924 for the simultaneous production of PHAs and rhamnolipids . High concentrations of rhamnolipids in the culture supernatant were attained at the end of both the first and second batch cultures . High PHA content was achieved in the resting cells that were finally harvested after the second batch . Simultaneous production of PHAs and rhamnolipids will enhance the availability of valuable biocatalysts of bacterial cells, and dispel the common belief that the production cost of PHAs accumulated intracellularly is almost impossible to become lower than that of cells themselves .

Crit Care Med, 2002 Mar, 30(3), 521 - 8
Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa; Hauser AR et al.; OBJECTIVE: Pseudomonas aeruginosa is a frequent cause of ventilator-associated pneumonia . Recent evidence suggests that production of type III secretion proteins is correlated with increased pathogenicity in both cellular and animal models of infection . The objective of this study was to determine whether this system contributes to disease severity in humans with ventilator-associated pneumonia . DESIGN: Retrospective pilot cohort study . SETTING: University hospital . PATIENTS: Thirty-five mechanically ventilated patients with bronchoscopically confirmed ventilator-associated pneumonia caused by P . aeruginosa . MEASUREMENTS AND MAIN RESULTS: Ventilator-associated pneumonia was categorized as severe (patients died or had a recurrence of their pneumonia despite appropriate antibiotic therapy) or mild (patients uneventfully recovered from their pneumonia) . The type III secretion genotypes and phenotypes of isolates cultured from the patients with ventilator-associated pneumonia were determined . Whereas every examined isolate harbored type III secretion genes, only 27 (77%) were capable of secreting detectable amounts of type III proteins in vitro . Twenty-two (81%) of the patients infected with these 27 isolates had severe disease . Of the eight isolates that did not secrete type III proteins, only three (38%) were cultured from patients with severe disease . Thus, infection with a type-III-secreting isolate correlated with severe disease (p < .05) . In vitro assays indicated that ExoU, the type III effector protein most closely linked to mortality in animal models, was secreted in detectable amounts in vitro by 10 (29%) of the 35 examined isolates . Nine (90%) of these 10 isolates were cultured from patients with severe disease (p < .05 when compared with the nonsecreting isolates) . In contrast, ExoS was secreted by 16 (46%) of the 35 examined isolates . Twelve (75%) of these 16 isolates were cultured from patients with severe disease (p = .14 when compared with the nonsecreting isolates) . CONCLUSIONS: In patients with ventilator-associated pneumonia, type-III-secreting isolates were associated with worse clinical outcomes, suggesting that this secretion system plays an important role in human disease . Our findings support the hypothesis that antibodies targeted against these proteins may be useful as adjunctive therapy in intubated patients with P . aeruginosa colonization or infection.

Microbiology, 2002 May, 148(Pt 5), 1561 - 9
Effect of vfr mutation on global gene expression and catabolite repression control of Pseudomonas aeruginosa; Suh SJ et al.; Vfr of Pseudomonas aeruginosa is 91% similar to the cAMP receptor protein (CRP) of Escherichia coli . Based on the high degree of sequence homology between the two proteins, the question arose whether Vfr had a global regulatory effect on gene expression for P . aeruginosa as CRP did for E . coli . This report provides two-dimensional polyacrylamide gel electrophoretic evidence that Vfr is a global regulator of gene expression in P . aeruginosa . In a vfr101::aacC1 null mutant, at least 43 protein spots were absent or decreased when compared to the proteome pattern of the parent strain . In contrast, 17 protein spots were absent or decreased in the parent strain when compared to the vfr101::aacC1 mutant . Thus, a mutation in vfr affected production of at least 60 proteins in P . aeruginosa . In addition, the question whether Vfr and CRP shared similar mechanistic characteristics was addressed . To ascertain whether Vfr, like CRP, can bind cAMP, Vfr and CRP were purified to homogeneity and their apparent dissociation constants (K(d)) for binding to cAMP were determined . The K(d) values were 1.6 microM for Vfr and 0.4 microM for CRP, suggesting that these proteins have a similar affinity for cAMP . Previously the authors had demonstrated that Vfr could complement a crp mutation and modulate catabolite repression in E . coli . This study presents evidence that Vfr binds to the E . coli lac promoter and that this binding requires the presence of cAMP . Finally, the possible involvement of Vfr in catabolite repression control in P . aeruginosa was investigated . It was found that succinate repressed production of mannitol dehydrogenase, glucose-6-phosphate dehydrogenase, amidase and urocanase both in the parent and in two vfr null mutants . This implied that catabolite repression control was not affected by the vfr null mutation . In support of this, the cloned vfr gene failed to complement a mutation in the P . aeruginosa crc gene . Thus, although Vfr is structurally similar to CRP, and is a global regulator of gene expression in P . aeruginosa, Vfr is not required for catabolite repression control in this bacterium.

Mol Microbiol, 2002 Jan, 43(2), 475 - 85
A novel type II secretion system in Pseudomonas aeruginosa; Ball G et al.; The genome sequence of Pseudomonas aeruginosa strain PAO1 has been determined to facilitate postgenomic studies aimed at understanding the capacity of adaptation of this ubiquitous opportunistic pathogen . P . aeruginosa produces toxins and hydrolytic enzymes that are secreted via the type II secretory pathway using the Xcp machinery or 'secreton' . In this study, we characterized a novel gene cluster, called hxc for homologous to xcp . Characterization of an hxcR mutant, grown in phosphate-limiting medium, revealed the absence of a 40 kDa protein found in the culture supernatant of wild-type or xcp derivative mutant strains . The protein corresponded to the alkaline phosphatase L-AP, renamed LapA, which is secreted in an xcp-independent but hxc-dependent manner . Finally, we showed that expression of the hxc gene cluster is under phosphate regulation . This is the first report of the existence of two functional type II secretory pathways within the same organism, which could be related to the high adaptation potential of P . aeruginosa.

Eur J Biochem, 2002 Apr, 269(8), 2194 - 203
Structural studies on the core and the O-polysaccharide repeating unit of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide; Bystrova OV et al.; The structure of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 1 was studied after mild acid and strong alkaline degradations by MS and NMR spectroscopy . Three types of LPS molecules were found, including those with an unsubstituted glycoform 1 core (A) or an isomeric glycoform 2 core substituted with one O-polysaccharide repeating unit (B) or with a long-chain O-polysaccharide . Therefore, of two core glycoforms, only glycoform 2 accepts the O-polysaccharide . In the structures A and B, Kdo, Hep, Hep7Cm, GalNAcAN3Ac, GalNFoAN, QuiNAc, GalNAla represent 3-deoxy-d-manno-octulosonic acid, l-glycero-d-manno-heptose, 7-O-carbamoyl-l-glycero-d-manno-heptose, 2-acetamido-3-O-acetyl-2-deoxygalacturonamide, 2-formamido-2-deoxygalacturonamide, 2-acetamido-2,6-dideoxyglucose and 2-(l-alanylamino)-2-deoxygalactose, respectively; all sugars are in the pyranose form and have the d configuration unless otherwise stated . One or more phosphorylation sites may be occupied by diphosphate groups . In a minority of the LPS molecules, an O-acetyl group is present in the outer core region at unknown position . The site and the configuration of the linkage between the O-polysaccharide and the core and the structure of the O-polysaccharide repeating unit were defined in P . aeruginosa immunotype 1 . The QuiNAc residue linked to the Rha residue of the core was found to have the beta configuration, whereas in the interior repeating units of the O-polysaccharide this residue is in the alpha-configuration . The data obtained are in accordance with the initiation of biosynthesis of the O-polysaccharide of P . aeruginosa O6, which is closely related to immunotype 1, by transfer of d-QuiNAc-1-P to undecaprenyl phosphate followed by synthesis of the repeating O-antigen tetrasaccharide.

J Endotoxin Res, 2002, 8(1), 39 - 46
The biological activity of a liposomal complete core lipopolysaccharide vaccine; Erridge C et al.; A vaccine that induces humoral immunity to lipopolysaccharide (LPS), while remaining non-pyrogenic should be beneficial, as high levels of antibodies against LPS are associated with a reduced risk of adverse outcome . However, pure LPS or bacteria expressing LPS are generally considered too toxic to be used as vaccines . Recently, a novel, immunogenic complete core lipopolysaccharide vaccine has been described, which has been designed to prevent endotoxin-related inflammatory reactions in surgical and high-risk hospitalized patients . In vivo studies have shown that while administration of the vaccine to rabbits results in no toxicity over 7 days, it does induce significantly enhanced antibody responses towards a broad range of clinically relevant Gram-negative LPSs . Here we show that encapsulation of the four complete core LPS types Escherichia coli K12, Escherichia coli R1, Bacteroides fragilis and Pseudomonas aeruginosa into liposomes greatly reduces the ability of a given amount of LPS to induce TNF-alpha production in vitro from human monocytes . In contrast to previous studies of liposomal LPS, we demonstrate a reduction in activity of approximately 100,000-fold; a reduction approximately 100-1,000-fold more than that previously described . The signalling by the liposomal LPS appears to be entirely dependent on serum factors, though this can be partially restored by soluble CD14 or, to a lesser extent, by lipopolysaccharide binding protein . Time-course experiments reveal that liposomal LPS signalling shows similar kinetics to pure LPS signalling . Therefore, as well as inducing specific antibody responses, liposomal LPS demonstrates characteristics suitable for use as a vaccine to be used in human beings.

Invest Ophthalmol Vis Sci, 2002 May, 43(5), 1437 - 44
Pseudomonas aeruginosa exotoxin A and keratitis in mice; Pillar CM et al.; PURPOSE: To determine the importance of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor in corneal disease . METHODS: Isogenic mutants deficient in ETA were constructed in P . aeruginosa strains PAO1 and ATCC 19660 by allelic exchange and then evaluated for virulence in a mouse model of bacterial keratitis . The effect of ETA on adherence to scarified corneal epithelium was assessed in an in vitro organ culture model . RESULTS: Mutants of either P . aeruginosa PAO1 or 19660, deficient in ETA, adhered to wounded corneal tissue and initiated ocular disease similar to that in wild-type strains . However, in contrast to wild-type strains, ETA mutants were quickly cleared from the eye, inflammation diminished, and the cornea healed . CONCLUSIONS: Although ETA has no effect on the ability of P . aeruginosa to adhere to corneal wounds or to initiate Pseudomonas keratitis, it is crucial for the organism to persist in the eye and ultimately cause disease.

Braz J Infect Dis, 2002 Feb, 6(1), 1 - 7
Antibiotic resistance and molecular typing of Pseudomonas aeruginosa: focus on imipenem; de Freitas AL et al.; Susceptibility tests by disk diffusion and by E-test and molecular typing by macrorestriction analysis were performed to determine the relatedness of Pseudomonas aeruginosa isolates from three distinct hospitals . The resistance profile of 124 isolates to 8 antimicrobial agents was determined in three different hospitals, in Porto Alegre, Brazil . Frequencies of susceptibility ranged from 43.9% for carbenicillin to 87.7% for ceftazidime . Cross-resistance data of imipenem-resistant isolates indicated that most (70%) were also resistant to carbenicillin, although 30% remained susceptible to ceftazidime and cefepime . In general, susceptibility profiles were not able to determine relatedness among isolates of P . aeruginosa . On the other hand, molecular typing by macrorestriction analysis demonstrated high discriminatory power and identified 66 strains among 72 isolates of P . aeruginosa . Imipenem-susceptible isolates were all different . However, identical clones of imipenem-resistant isolates were found in two of the hospitals, despite variable response to other antibiotics . No clustering of infection among the different medical centers was observed . In conclusion, clones of P . aeruginosa did not spread among the different hospitals in our city even though related isolates of imipenem-resistant P . aeruginosa were found.

Hum Mol Genet, 2002 May 1, 11(9), 1059 - 67
Gene complementation of airway epithelium in the cystic fibrosis mouse is necessary and sufficient to correct the pathogen clearance and inflammatory abnormalities; Oceandy D et al.; Increasingly, cystic fibrosis (CF) is regarded as an inflammatory disorder where the response of the lung to Pseudomonas aeruginosa is exaggerated as a consequence of processes mediated by the product of the CF gene, CFTR . Of importance to any gene-replacement strategy for treatment of CF is the identification of the cell type(s) within the lung milieu that need to be corrected and an indication whether this is sufficient to restore a normal inflammatory response and bacterial clearance . We generated G551D CF mice transgenically expressing the human CFTR gene in two tissue compartments previously demonstrated to mediate a CFTR-dependent inflammatory response: lung epithelium and alveolar macrophages . Following chronic pulmonary infection with P . aeruginosa, CF mice with epithelial-expressed but not macrophage-specific CFTR showed an improvement in pathogen clearance and inflammatory markers compared with control CF animals . Additionally, these data indicate the general role for epithelial cell-mediated events in the response of the lung to bacterial pathogens and the importance of CFTR in mediating these processes.

Arch Dis Child Fetal Neonatal Ed, 2002 May, 86(3), F204 - 6
Exogenous Pseudomonas endophthalmitis: a cause of lens enucleation; Gaili H et al.; Pseudomonas aeruginosa eye infection, although uncommon, may be a devastating disease if not recognised and treated appropriately, especially in premature infants . The case is presented of a premature baby who lost her right eye from invasive exogenous Ps aeruginosa eye infection.

Eur J Dermatol, 2002 May-Jun, 12(3), 291 - 2
Guess what! Pseudomonas aeruginosa sepsis; Bugatti L et al.; A 62-year-old woman affected by end-stage renal disease secondary to Waldenstrom's disease was admitted to place a central venous catheter for hemodialysis purposes . During the admission, she gradually developed a number of necrotic ulcerative and fluctuant nodular skin lesions on the submammary flexures, groins and limbs accompanied by high fever and chills . Yellow-green purulent material could be drained from the site of introduction of the jugular catheter . Skin biopsies were taken from the edge of an inguinal necrotic-ulcerative lesion and from a fluctuant nodular lesion of the thigh, where pus was drained and cultured.

Jpn J Antibiot, 2002 Feb, 55(1), 67 - 76
{Bactericidal activity of biapenem against various efflux system mutants of Pseudomonas aeruginosa}; Hiraishi T et al.; The bactericidal activity of biapenem, a new carbapenem, against various efflux-mutants of Pseudomonas aeruginosa was compared with those of imipenem, panipenem, meropenem and ceftazidime . The bactericidal activity of biapenem against P . aeruginosa KG5001, a strain deficient in MexAB-OprM, MexCD-OprJ and MexXY-OprM, was very strong compared with those of imipenem and meropenem . In terms of bactericidal activities, biapenem and imipenem had similar activities against P . aeruginosa KG5003, a strain overexpressing MexAB-OprM, as against P . aeruginosa KG5001, however meropenem and ceftazidime had weaker activities against KG5003 than KG5001 . The bactericidal activity against P . aeruginosa KG5007, a strain overexpressing MexCD-OprJ, was observed only by biapenem . The bactericidal activity of biapenem was strong and not influenced by all of these three efflux systems.

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