Pseudomonas aeruginosa

Medicine (Baltimore), 2004 May, 83(3), 139 - 48
Sternoclavicular septic arthritis: review of 180 cases; Ross JJ et al.; We review 170 previously reported cases of sternoclavicular septic arthritis, and report 10 new cases . The mean age of patients was 45 years; 73% were male . Patients presented with chest pain (78%) and shoulder pain (24%) after a median duration of symptoms of 14 days . Only 65% were febrile . Bacteremia was present in 62% . Common risk factors included intravenous drug use (21%), distant site of infection (15%), diabetes mellitus (13%), trauma (12%), and infected central venous line (9%) . No risk factor was found in 23% . Serious complications such as osteomyelitis (55%), chest wall abscess or phlegmon (25%), and mediastinitis (13%) were common . Staphylococcus aureus was responsible for 49% of cases, and is now the major cause of sternoclavicular septic arthritis in intravenous drug users . Pseudomonas aeruginosa infection in injection drug users declined dramatically with the end of an epidemic of pentazocine abuse in the 1980s . Sternoclavicular septic arthritis accounts for 1% of septic arthritis in the general population, but 17% in intravenous drug users, for unclear reasons . Bacteria may enter the sternoclavicular joint from the adjacent valves of the subclavian vein after injection of contaminated drugs into the upper extremity, or the joint may become infected after attempted drug injection between the heads of the sternocleidomastoid muscle . Computed tomography or magnetic resonance imaging should be obtained routinely to assess for the presence of chest wall phlegmon, retrosternal abscess, or mediastinitis . If present, en-bloc resection of the sternoclavicular joint is indicated, possibly with ipsilateral pectoralis major muscle flap . Empiric antibiotic therapy may need to cover methicillin-resistant Staphylococcus aureus (MRSA).

J Biol Chem, 2004 Jun 18, 279(25), 25939 - 42 Epub 2004 Apr 26.
Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa; Akama H et al.; The MexAB-OprM efflux pump of Pseudomonas aeruginosa is central to multidrug resistance of this organism, which infects immunocompromised hospital patients . The MexA, MexB, and OprM subunits were assumed to function as the membrane fusion protein, the body of the transporter, and the outer membrane channel protein, respectively . For better understanding of this important xenobiotic transporter, we show the x-ray crystallographic structure of MexA at a resolution of 2.40 A . The global MexA structure showed unforeseen new features with a spiral assembly of six and seven protomers that were joined together at one end by a pseudo 2-fold image . The protomer showed a new protein structure with a tandem arrangement consisting of at least three domains and presumably one more . The rod domain had a long hairpin of twisted coiled-coil that extended to one end . The second domain adjacent to the rod alpha-helical domain was globular and constructed by a cluster of eight short beta-sheets . The third domain located distal to the alpha-helical rod was globular and composed of seven short beta-sheets and one short alpha-helix . The 13-mer was shaped like a woven rattan cylinder with a large internal tubular space and widely opened flared ends . The 6-mer and 7-mer had a funnel-like structure consisting of a tubular rod at one side and a widely opened flared funnel top at the other side . Based on these results, we constructed a model of the MexAB-OprM pump assembly . The three pairs of MexA dimers interacted with the periplasmic alpha-barrel domain of OprM via the alpha-helical hairpin, the second domain interacted with both MexB and OprM at their contact site, and the third and disordered domains probably interacted with the distal domain of MexB . In this fashion, the MexA subunit connected MexB and OprM, indicating that MexA is the membrane bridge protein.

J Infect Dis, 2004 May 1, 189(9), 1590 - 7 Epub 2004 Apr 16.
Relationship between fluoroquinolone area under the curve: minimum inhibitory concentration ratio and the probability of eradication of the infecting pathogen, in patients with nosocomial pneumonia; Drusano GL et al.; Our objective was to prospectively determine the factors influencing the probability of a good microbiological or clinical outcome in patients with nosocomial pneumonia treated with a fluoroquinolone . Levofloxacin was administered as an infusion of 500 mg/h for 1.5 h (total dose, 750 mg) . For patients with Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus, a second drug was added (ceftazidime or piperacillin/tazobactam for P . aeruginosa and vancomycin for methicillin-resistant S . aureus) . Population pharmacokinetic studies of 58 patients demonstrated that this population handled the drug differently from populations of volunteers . Multivariate logistic regression analysis (n=47 patients) demonstrated that only the age of the patient and the achievement of an area under the curve: minimum inhibitory concentration ratio of > or =87 had a significant effect on eradication of the pathogen (P<.001) . Achieving the breakpoint made the patient 4 times more likely to achieve eradication . The effect was greatest in patients > or =67 years old.

Photochem Photobiol, 2004 Mar, 79(3), 297 - 302
Effect of monovalent and divalent cations on the photoinactivation of bacteria with meso-substituted cationic porphyrins; Lambrechts SA et al.; It is well established that for successful photoinactivation (PI) of gram-negative bacteria a cationic photosensitizer is required . This requirement suggests a charge-dependent interaction between the photosensitizer and the gram-negative bacterium, which may be influenced by the presence of ions in the suspending medium . The aim of the present study was to investigate the effect of cations Na+ and Ca2+ on the efficacy of the PI of the gram-negative Pseudomonas aeruginosa and the gram-positive Staphylococcus aureus . The bacteria were suspended in buffer containing either meso-tetra(N-methyl-4-pyridyl)-porphyrin or meso-mono-phenyl-tri(N-methyl-4-pyridyl)-porphyrin as photosensitizer and various concentrations of Na+ or Ca2+ . The cell suspensions were exposed to a broadband light dose of 9 J/cm2 . In buffer without added cations, P . aeruginosa and S . aureus were equally sensitive to PI . Addition of cations strongly decreased the sensitivity of both bacteria to PI, with the PI of P . aeruginosa being much more decreased than that of S . aureus, and Ca2+ being more effective than Na+ . The decreased sensitivity was accompanied by a reduced binding of the photosensitizers to the bacteria.

Anal Biochem, 2004 May 15, 328(2), 225 - 32
A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction; Kessler E et al.; Pseudomonas aeruginosa LasA protease is a secreted metalloendopeptidase that can lyse Staphylococcus aureus cells by cleaving the pentaglycine bridges of their peptidoglycan . It can also degrade elastin and stimulate shedding of cell-surface proteoglycans, activities implicated in pathogenesis of P . aeruginosa infections . The activity of LasA protease can be assayed spectrophotometrically by following the reduction in turbidity of S . aureus cell suspensions . This assay, however, does not permit kinetic studies and its reproducibility is poor . Here we describe a two-stage enzymatic reaction for the continuous measurement of LasA protease activity using a defined substrate, succinyl-Gly-Gly-Phe-4-nitroanilide, supplemented with Streptomyces griseus aminopeptidase . Cleavage of the Gly-Phe bond by LasA protease is followed by hydrolysis of the product Phe-4-nitroanilide by the aminopeptidase and the rate of release of the chromophore (4-nitroaniline) is measured spectrophotometrically using a 96-well microplate reader . Activity of nanogram amounts of LasA protease could be determined within a few minutes . Furthermore, this assay permitted the determination of Km and kcat values for LasA protease, which were 0.46 mM and 11.8s(-1), respectively . Pseudomonas elastase was also active in the assay . However, it was less effective than LasA protease and its activity was inhibited by phosphoramidon . The assay is highly sensitive and reproducible, providing a convenient tool for further studies of LasA protease function(s) and mechanism of action.

Clin Microbiol Infect, 2004 May, 10(5), 431 - 5
Intravenous catheter infections associated with bacteraemia: a 2-year study in a university hospital; Paragioudaki M et al.; The aim of this retrospective study was to assess the incidence and aetiology of central and peripheral venous catheter (C/PVC) infections during a 2-year period (1999-2000) and to determine the susceptibility of isolated microorganisms to various antimicrobial agents . Catheter tips were processed using the semiquantitative method and blood cultures were performed with the BacT/Alert automated system . Antibiotic susceptibilities were performed by disk agar diffusion and MICs were determined by Etest, according to NCCLS standards . During the study period, samples from 1039 C/PVC infections were evaluated, yielding 384 (37.0%) positive cultures . Blood cultures were also available from 274 patients, of which 155 (56.6%) yielded the same microorganism as from the catheter . No bloodstream infections were detected in 104 C/PVC-positive cases . Methicillin-resistant coagulase-negative staphylococci were the most frequent isolates, followed by Gram-negative bacteria, especially Pseudomonas aeruginosa . Resistance to glycopeptides among staphylococci and enterococci was not detected, whereas 60% of Gram-negative bacilli were resistant to beta-lactams.

Exp Eye Res, 2004 Jun, 78(6), 1155 - 62
TIMP-1 role in protection against Pseudomonas aeruginosa-induced corneal destruction; Kernacki KA et al.; To establish the role of TIMP-1 in protection against Pseudomonas aeruginosa-induced corneal destruction, corneas of adult 8-week-old (resistant) and aged 12-month-old (susceptible) mice were infected with the bacterium . Corneas were analyzed for TIMP-1 protein by immunocytochemistry and Western blotting . Basement membrane (BM) integrity was assessed by immunostaining for type IV collagen . Additionally, resistant 8-week-old mice were treated systemically with neutralizing TIMP-1 polyclonal antibody (pAb) or pre-immune normal rabbit serum (NRS) . Ocular and BM integrity as well as MMP-9 expression were examined in these mice . A greater amount of TIMP-1 protein was observed in the cornea of 8-week-old mice . In the cornea, the strongest staining was found in the superficial epithelium, but positive staining also was seen in the basal epithelium and stroma . When type IV collagen was analyzed in the BM of both age groups of mice, a distinct staining pattern was observed in only the young adult mice . Treatment of 8-week-old resistant mice with neutralizing TIMP-1 pAb vs NRS increased the amount of MMP-9 in the cornea of TIMP-1 pAb-treated mice and affected the ability of these mice to deposit BM components . These studies suggest that adequate expression of TIMP-1 protects against BM and stromal degradation via multiple processes.

Bioorg Med Chem Lett, 2004 May 17, 14(10), 2493 - 7
MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa . Part 4: Addressing the problem of poor stability due to photoisomerization of an acrylic acid moiety; Nakayama K et al.; Exchange of the ethylene tether in a series of pyridopyrimidine-based MexAB-OprM specific efflux pump inhibitors to an amide bond stabilized the olefin of the acrylic acid moiety, preventing facile photoisomerization to the Z-isomer . Furthermore, the activity was drastically improved in the amide tether variants, providing extremely potent acrylic acid and vinyl tetrazole analogues.

Emerg Infect Dis, 2004 Mar, 10(3), 535 - 8
Endemic carbapenem-resistant Pseudomonas aeruginosa with acquired metallo-beta-lactamase determinants in European hospital; Lagatolla C et al.; Acquired metallo-beta-lactamases (MBLs) can confer broad-spectrum beta-lactam resistance (including carbapenems) not reversible by conventional beta-lactamase inhibitors and are emerging resistance determinants of remarkable clinical importance . In 2001, multidrug-resistant Pseudomonas aeruginosa carrying bla(VIM) MBL genes were found to be widespread (approximately 20% of all P . aeruginosa isolates and 70% of the carbapenem-resistant isolates) at Trieste University Hospital . Clonal diversity and heterogeneity of resistance determinants (either bla(VIM-1)-like or bla(VIM-2)-like) were detected among MBL producers . This evidence is the first that acquired MBLs can rapidly emerge and establish a condition of endemicity in certain epidemiologic settings.

Am J Physiol Lung Cell Mol Physiol, 2004 Aug, 287(2), L402 - 10 Epub 2004 Apr 23.
Effect of lung-protective ventilation on severe Pseudomonas aeruginosa pneumonia and sepsis in rats; Kurahashi K et al.; Pneumonia caused by Pseudomonas aeruginosa carries a high rate of morbidity and mortality . A lung-protective strategy using low tidal volume (V(T)) ventilation for acute lung injury improves patient outcomes . The goal of this study was to determine whether low V(T) ventilation has similar utility in severe P . aeruginosa infection . A cytotoxic P . aeruginosa strain, PA103, was instilled into the left lung of rats anesthetized with pentobarbital . The lung-protective effect of low V(T) (6 ml/kg) with or without high positive end-expiratory pressure (PEEP, 10 or 3 cmH(2)O) was then compared with high V(T) with low PEEP ventilation (V(T) 12 ml/kg, PEEP 3 cmH(2)O) . Severe lung injury and septic shock was induced . Although ventilatory mode had little effect on the involved lung or septic physiology, injury to noninvolved regions was attenuated by low V(T) ventilation as indicated by the wet-to-dry weight ratio (W/D; 6.13 +/- 0.78 vs . 3.78 +/- 0.26, respectively) and confirmed by histopathological examinations . High PEEP did not yield a significant protective effect (W/D, 4.03 +/- 0.32) but, rather, caused overdistension of noninvolved lungs . Bronchoalveolar lavage revealed higher concentrations of TNF-alpha in the fluid of noninvolved lung undergoing high V(T) ventilation compared with those animals receiving low V(T) . We conclude that low V(T) ventilation is protective in noninvolved regions and that the application of high PEEP attenuated the beneficial effects of low V(T) ventilation, at least short term . Furthermore, low V(T) ventilation cannot protect the involved lung, and high PEEP did not significantly alter lung injury over a short time course.

Cochrane Database Syst Rev . 2004;(2):CD002203.
Macrolide antibiotics for cystic fibrosis; Southern KW et al.; BACKGROUND: Chronic severe infection with Pseudomonas aeruginosa, affects many people with cystic fibrosis (CF) . There is evidence from the laboratory and from other disease processes that macrolide antibiotics, whilst not directly active against Pseudomonas aeruginosa, may have indirect actions against this organism . OBJECTIVES: We aimed to test the hypotheses that, in people with CF, macrolide antibiotics:(1) improve clinical status compared to placebo or another antibiotic;(2) do not have unacceptable adverse effects.If benefit was demonstrated, we aimed to assess the optimal type, dose and duration of macrolide therapy . SEARCH STRATEGY: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group trials register comprising references identified from comprehensive electronic database searches, handsearching relevant journals and abstract books of conference proceedings.We contacted principal investigators known to work in the field, previous authors and pharmaceutical companies who manufacture macrolide antibiotics for unpublished or follow-up data (December 2003).Most recent search of the Group's register: January 2004 SELECTION CRITERIA: Published or unpublished randomised controlled trials of macrolide antibiotics compared to placebo, another class of antibiotic or another macrolide antibiotic . Studies comparing regimens of the same macrolide antibiotic at different doses will also be included . DATA COLLECTION AND ANALYSIS: Two reviewers independently extracted data and assessed study quality . Three groups were contacted for missing data and we hope to include these in future reviews . MAIN RESULTS: Searches identified 14 studies, four were included in this review (296 participants) . Two studies enrolled adults, one children (a significant number of whom were not colonised with Pseudomonas aeruginosa) and one both adults and children . All the clinical studies reported small but significant improvements in respiratory function with azithromycin versus placebo . Meta-analysis at the one-month and six-month time points demonstrates a significant benefit with respect to relative change in FEV1 (at six months, for n = 104, azithromycin and n = 114, placebo; WMD 5.82% (95% CI 2.45 to 9.20)) . The largest study reported a significant increase in mild adverse events (nausea, diarrhoea and wheezing) . REVIEWERS' CONCLUSIONS: There is clear evidence from these studies of a small but significant improvement in respiratory function following treatment with azithromycin . The largest study employed a three times a week dose and, in this study, treatment with azithromycin was associated with a significant increase in mild adverse events . Further studies are needed to clarify the precise role of azithromycin in the treatment of CF lung disease.

Antimicrob Agents Chemother, 2004 May, 48(5), 1879 - 81
Rapid colorimetric assay for antimicrobial susceptibility testing of Pseudomonas aeruginosa; Tunney MM et al.; A colorimetric assay based on the reduction of a tetrazolium salt {2,3-bis{2-methyloxy-4-nitro-5-sulfophenyl}-2H-tetrazolium-5-carboxanilide (XTT)} for rapidly determining the susceptibility of Pseudomonas aeruginosa isolates to bactericidal antibiotics is described . There was excellent agreement between the tobramycin and ofloxacin MICs determined after 5 h using the XTT assay and after 18 h using conventional methods . The data suggests that an XTT-based assay could provide a useful method for rapidly determining the susceptibility of P . aeruginosa to bactericidal antibiotics.

Antimicrob Agents Chemother, 2004 May, 48(5), 1876 - 8
Virulence of metallo-beta-lactamase-producing Pseudomonas aeruginosa in vitro and in vivo; Aoki S et al.; We evaluated the virulence of Pseudomonas aeruginosa carrying bla(IMP), a metallo-beta-lactamase gene, and the efficacy of ceftazidime, imipenem-cilastatin, and ciprofloxacin in the endogenous bacteremia model . The presence of bla(IMP) did not practically change the virulence of the parent strain, and ciprofloxacin was effective against infection with P . aeruginosa carrying bla(IMP).

Antimicrob Agents Chemother, 2004 May, 48(5), 1797 - 802
Clinical strains of Pseudomonas aeruginosa overproducing MexAB-OprM and MexXY efflux pumps simultaneously; Llanes C et al.; Simultaneous overexpression of the MexAB-OprM and MexXY efflux systems was demonstrated by real-time reverse transcription-PCR and immunoblotting experiments for 12 multiresistant clinical isolates of Pseudomonas aeruginosa . DNA sequencing analysis showed that nine of these strains (named agrZ mutants) harbored mutations in mexZ, the product of which downregulates the expression of the mexXY operon . In addition, 8 of the 12 strains exhibited mutations in genes known to control transcription of the mexAB-oprM operon . Four of them were nalB mutants with alterations in the repressor gene mexR, three of them appeared to be nalC mutants deficient in gene PA3721 and overexpressing gene PA3720, and one strain was a nalB nalC double mutant . For MexAB-OprM as well as for MexXY, no clear correlation could be established between (i) the types of mutations, (ii) the expression level of mexA or mexX, and (iii) resistance to effluxed antibiotics . Finally, three isolates, named agrW mutants, overproduced MexXY and had an intact mexZ gene, and four strains overproduced MexAB-OprM and had intact mexR and PA3721 genes (nalD mutants) . These data show that clinical isolates are able to broaden their drug resistance profiles by coexpressing two Mex efflux pumps and suggest the existence of additional regulators for MexAB-OprM and MexXY.

Antimicrob Agents Chemother, 2004 May, 48(5), 1681 - 7
Pseudomonas aeruginosa LasA protease in treatment of experimental staphylococcal keratitis; Barequet IS et al.; LasA protease is a staphylolytic endopeptidase secreted by Pseudomonas aeruginosa . We have examined the effectiveness of LasA protease in the treatment of staphylococcal keratitis caused by methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S . aureus (MRSA) isolates in a rabbit model . Keratitis was induced by intrastromal injection of the bacteria . The eyes were treated topically, and the efficacy of LasA protease was compared to those of lysostaphin (a staphylolytic protease secreted by Staphylococcus simulans) and vancomycin . When treatment was initiated early (4 h) after infection, practically all of the MSSA- and MRSA-infected corneas were sterilized by LasA protease, and its efficacy in eradicating the bacteria was comparable to those of lysostaphin and vancomycin . By contrast, most of the control corneas were heavily infected, with median values of 4.5 x 10(6) (MSSA) and 5 x 10(5) (MRSA) CFU/cornea (P < 0.001) . When treatment was initiated late (10 h) after infection, LasA protease reduced the numbers of CFU in both MSSA- and MRSA-infected corneas by 3 to 4 orders of magnitude compared to the numbers of CFU for the controls (median values, 1,380 and 30 CFU/cornea, respectively, for the treated animals compared to 1.2 x 10(6) and 5 x 10(5) CFU/cornea for the respective controls {P = 0.001}), and it was more effective than vancomycin in eradicating MRSA cells (P = 0.02) . In both the early- and the late-treatment protocols, the clinical scores for eyes treated with LasA protease were significantly lower than those for the eyes of the corresponding controls and comparable to those for the lysostaphin- and vancomycin-treated eyes . We conclude that LasA protease is effective in the treatment of experimental S . aureus keratitis in rabbits and may have potential for the treatment of disease in humans.

Antimicrob Agents Chemother, 2004 May, 48(5), 1676 - 80
Role of the multidrug efflux system MexXY in the emergence of moderate resistance to aminoglycosides among Pseudomonas aeruginosa isolates from patients with cystic fibrosis; Vogne C et al.; This study investigates the role of active efflux system MexXY in the emergence of aminoglycoside (AG) resistance among cystic fibrosis (CF) isolates of Pseudomonas aeruginosa . Three genotypically related susceptible and resistant (S/R) bacterial pairs and three other AG-resistant CF strains were compared to four non-CF strains moderately resistant to AGs . As demonstrated by immunoblot experiments, pump MexY was strongly overproduced in all of the resistant bacteria . This MexXY upregulation was associated with a 2- to 16-fold increase in the MICs of AGs in the S/R pairs and lower intracellular accumulation of dihydrostreptomycin . Alterations in mexZ, the repressor gene of operon mexXY, were found in all of the AG-resistant CF isolates and in one non-CF strain . Complementation of these bacteria with a plasmid-borne mexZ gene dramatically reduced the MICs of AGs, thus highlighting the role played by MexXY in the development of moderate resistance in CF patients . In contrast, complementation of the three non-CF strains showing wild-type mexZ genes left residual levels of resistance to AGs . These data indicate that a locus different from mexZ may be involved in overproduction of MexXY and that other nonenzymatic mechanisms contribute to AG resistance in P . aeruginosa.

Cell Microbiol, 2004 Jun, 6(6), 521 - 33
Role of the cystic fibrosis transmembrane conductance regulator in internalization of Pseudomonas aeruginosa by polarized respiratory epithelial cells; Darling KE et al.; Pseudomonas aeruginosa is an important human pathogen, producing lung infection in individuals with cystic fibrosis (CF), patients who are ventilated and those who are neutropenic . The respiratory epithelium provides the initial barrier to infection . Pseudomonas aeruginosa can enter epithelial cells, although the mechanism of entry and the role of intracellular organisms in its life cycle are unclear . We devised a model of infection of polarized human respiratory epithelial cells with P . aeruginosa and investigated the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in adherence, uptake and IL-8 production by human respiratory epithelial cells . We found that a number of P . aeruginosa strains could invade and replicate within cells derived from a patient with CF . Intracellular bacteria did not produce host cell cytotoxicity over a period of 24 h . When these cells were transfected with wild-type CFTR, uptake of bacteria was significantly reduced and release of IL-8 following infection enhanced . We propose that internalized P . aeruginosa may play an important role in the pathogenesis of infection and that, by allowing greater internalization into epithelial cells, mutant CFTR results in an increased susceptibility of bronchial infection with this microbe.

Crit Rev Eukaryot Gene Expr, 2004, 14(1-2), 53 - 64
SCE jumping: genetic tool for allelic exchange in bacteria; Wong SM; As more microbial genome sequence information becomes available, the field of bacterial pathogenesis would benefit from the development of new genetic tools designed to facilitate gene function studies on a genomic scale . The complete DNA sequence of the bacterium Pseudomonas aeruginosa provides an opportunity to apply functional genomics to a major opportunistic human pathogen . Here, I describe the development of a new gene replacement scheme termed "SCE jumping" in P . aeruginosa . The system uses the yeast I-SceI homing endonuclease in conjunction with in vitro mariner-transposon mutagenesis to generate mutations within targeted regions of the chromosome for genetic footprinting . Use of SCE jumping for generating transposon insertion mutants is anticipated to be widely applicable to other bacterial organisms . This allelic exchange strategy is discussed in context with other methods of gene replacement strategies available in P . aeruginosa . Development of SCE jumping provides an excellent example of the power of importing systems from unrelated organisms to circumvent practical challenges in molecular genetics.

Ulus Travma Derg, 2004 Apr, 10(2), 110 - 4
{Clinical evaluation and treatment results of 30 patients with necrotizing fasciitis}; Ozgenel GY et al.; BACKGROUND: We retrospectively evaluated patients who underwent treatment for necrotizing fasciitis within a five-year period . METHODS: Thirty patients (4 females, 26 males; mean age 55 years; range 19 to 78 years) with necrotizing fasciitis were evaluated with respect to age, sex, etiology, predisposing factors, localization of infections, culture results, and treatment methods and results . RESULTS: The most common etiologic and predisposing factors were anorectal lesions (36.7%) and diabetes (53.3%), respectively . Wound cultures yielded Pseudomonas aeruginosa in 50% of the patients . Two strains of aerobic bacteria were isolated in three patients . All patients underwent extensive surgical debridement and received antibiotic therapy . Twenty-nine patients (96.6%) required more than one debridement, with a mean of 4.5 debridements . The ensuing skin defects following debridement were reconstructed with grafts or local flaps . No complications were encountered in the postoperative period . CONCLUSION: Early diagnosis and treatment result in decreased morbidity and prevent mortality in necrotizing fasciitis.

Acta Crystallogr D Biol Crystallogr, 2004 May, 60(Pt 5), 948 - 9 Epub 2004 Apr 21.
Crystallization and preliminary X-ray crystallographic analysis of nicotinic acid mononucleotide adenylyltransferase from Pseudomonas aeruginosa; Kim HL et al.; The enzyme nicotinic acid mononucleotide adenylyltransferase (NaMN AT; EC 2.7.7.18) is essential for the synthesis of nicotinamide adenine dinucleotide and is a potential target for antibiotics . It catalyzes the transfer of an adenyl group from ATP to nicotinic acid mononucleotide to form nicotinic acid adenine dinucleotide . NaMN AT from Pseudomonas aeruginosa was overexpressed in Escherichia coli and crystallized at 291 K using 100 mM bis-Tris propane pH 7.0, 700 mM trisodium citrate and 15%(v/v) glycerol . X-ray diffraction data have been collected to 1.70 A . The crystals are tetragonal, belonging to space group P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 65.02, c = 109.80 A . The presence of one monomer in the asymmetric unit gives a reasonable V(M) of 2.15 A(3) Da(-1), with a solvent content of 42.7%.

Infect Immun, 2004 May, 72(5), 2899 - 906
The alternative activation pathway and complement component C3 are critical for a protective immune response against Pseudomonas aeruginosa in a murine model of pneumonia; Mueller-Ortiz SL et al.; Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia, and approximately 80% of patients with cystic fibrosis are infected with this bacterium . To investigate the overall role of complement and the complement activation pathways in the host defense against P . aeruginosa pulmonary infection, we challenged C3-, C4-, and factor B-deficient mice with P . aeruginosa via intranasal inoculation . In these studies, C3(-/-) mice had a higher mortality rate than C3(+/+) mice . Factor B(-/-) mice, but not C4(-/-) mice, infected with P . aeruginosa had a mortality rate similar to that of C3(-/-) mice, indicating that in this model the alternative pathway of complement activation is required for the host defense against Pseudomonas infection . C3(-/-) mice had 6- to 7-fold more bacteria in the lungs and 48-fold more bacteria in the blood than did C3(+/+) mice at 24 h postinfection . In vitro, phagocytic cells from C3(+/+) or C3(-/-) mice exhibited a decreased ability to bind and/or ingest P . aeruginosa in the presence of C3-deficient serum compared to phagocytic cells in the presence of serum with sufficient C3 . C3(-/-) mice displayed a significant increase in neutrophils in the lungs and had higher levels of interleukin-1beta (IL-1beta), IL-6, IL-10, KC, and MIP-2 in the lungs at 24 h postinfection than did C3(+/+) mice . Collectively, these results indicate that complement activation by the alternative pathway is critical for the survival of mice infected with P . aeruginosa and that the protection provided by complement is at least in part due to C3-mediated opsonization and phagocytosis of P . aeruginosa.

BMC Struct Biol . 2004 Mar 08;4(1):5.
Crystal structure of the YffB protein from Pseudomonas aeruginosa suggests a glutathione-dependent thiol reductase function; Teplyakov A et al.; BACKGROUND: The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli . The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein . RESULTS: The structure was determined at 1.0 A resolution by single-wavelength anomalous diffraction . The fold is very similar to that of arsenate reductase, which is an extension of the thioredoxin fold . CONCLUSION: Given the conservation of the functionally important residues and the ability to bind glutathione, YffB is likely to function as a GSH-dependent thiol reductase.

Mol Microbiol, 2004 May, 52(3), 873 - 93
Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa; Whitchurch CB et al.; Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility . Twitching motility in P . aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK . Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved . The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine- and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its C-terminus, and as such represents one of the most complex signalling proteins yet described in nature . We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA . The Chp system is also required for full virulence in a mouse model of acute pneumonia.

Mol Microbiol, 2004 May, 52(3), 691 - 700
A new Ralstonia solanacearum high-affinity mannose-binding lectin RS-IIL structurally resembling the Pseudomonas aeruginosa fucose-specific lectin PA-IIL; Sudakevitz D et al.; The plant pathogen Ralstonia solanacearum produces two lectins, each with different affinity to fucose . We described previously the properties and sequence of the first lectin, RSL (subunit M(r) 9.9 kDa), which is related to fungal lectins (Sudakevitz, D., Imberty, A., and Gilboa-Garber, N., 2002, J Biochem 132: 353-358) . The present communication reports the discovery of the second one, RS-IIL (subunit M(r) 11.6 kDa), a tetrameric lectin, with high sequence similarity to the fucose-binding lectin PA-IIL of Pseudomonas aeruginosa . RS-IIL recognizes fucose but displays much higher affinity to mannose and fructose, which is opposite to the preference spectrum of PA-IIL . Determination of the crystal structure of RS-IIL complexed with a mannose derivative demonstrates a tetrameric structure very similar to the recently solved PA-IIL structure (Mitchell, E., et al., 2002, Nature Struct Biol 9: 918-921) . Each monomer contains two close calcium cations that mediate the binding of the monosaccharide and explain the outstandingly high affinity to the monosaccharide ligand . The binding loop of the cations is fully conserved in RS-IIL and PA-IIL, whereas the preference for mannose versus fucose can be attributed to the change of a three-amino-acid sequence in the 'specificity loop'.

Otolaryngol Head Neck Surg, 2004 Apr, 130(4), 430 - 6
Hearing loss with semicircular canal fistula in exotoxin A-deficient Pseudomonas otitis media; Yunus TM et al.; OBJECTIVE: The study goal was to compare the severity of hearing loss with semicircular canal injury in the presence of otitis media (OM) due to an exotoxin A-producing strain of Pseudomonas aeruginosa (PA+) and an exotoxin A-deficient daughter strain (PA-) . METHODS: PA+ or PA- was injected bilaterally into guinea pig ears . Three days later, unilateral lateral semicircular canal transection was performed . Hearing was tested before and after transection . RESULTS: PA OM was induced in all injected ears . Significant elevation of click and 12-kHz thresholds (P < 0.0001) were encountered in PA+-infected ears . No significant threshold elevations were encountered in PA--infected ears . CONCLUSION: Hearing loss resulting from canal injury in the presence of Pseudomonas OM may be mediated by exotoxin A . SIGNIFICANCE: Treatment to neutralize the effects of exotoxin A may minimize the risk of hearing loss with canal fistula in chronic OM.

Biochim Biophys Acta, 2004 Apr 12, 1655(1-3), 59 - 63
Electron tunneling in rhenium-modified Pseudomonas aeruginosa azurins; Miller JE et al.; Laser flash-quench methods have been used to generate tyrosine and tryptophan radicals in structurally characterized rhenium-modified Pseudomonas aeruginosa azurins . Cu(I) to "Re(II)" electron tunneling in Re(H107) azurin occurs in the microsecond range . This reaction is much faster than that studied previously for Cu(I) to Ru(III) tunneling in Ru(H107) azurin, suggesting that a multistep ("hopping") mechanism might be involved . Although a Y108 radical can be generated by flash-quenching a Re(H107)M(II) (M=Cu, Zn) protein, the evidence suggests that it is not an active intermediate in the enhanced Cu(I) oxidation . Rather, the likely explanation is rapid conversion of Re(II)(H107) to deprotonated Re(I)(H107 radical), followed by electron tunneling from Cu(I) to the hole in the imidazole ligand.

J Ethnopharmacol, 2004 May, 92(1), 135 - 44
Evaluation of extracts of Anthocleista djalonensis, Nauclea latifolia and Uvaria afzalii for activity against bacterial isolates from cases of non-gonococcal urethritis; Okoli AS et al.; Whole root preparations of three Nigerian medicinal plants, Anthocleista djalonensis, Nauclea latifolia and Uvaria afzalii, used traditionally in combination treatment of sexually transmitted diseases (STD), were extracted by maceration in ethanol, cold and hot water, respectively . The extracts were tested, by agar diffusion and macrobroth dilution methods, for activity against five strains of Staphylococcus aureus and two of Escherichia coli isolated from cases of STD and or urethritis . Four typed bacterial strains, S aureus ATCC 12600, Bacillus subtilis ATCC 6051, Pseudomonas aeruginosa ATCC 10145 and Escherichia coli ATCC 117755 were included as reference organisms . Ethanolic and cold-water extracts of Anthocliesta djalonensis exhibited activity against 9 and 7, respectively, of the 11 test organisms . They were bacteriostatic at minimum inhibitory concentrations (MIC) to the Gram positive strains but bactericidal to the Gram negative strains . Similar crude extracts of Uvaria afzalii showed bactericidal activity restricted to Gram positive (Staphylococcus aureus and Bacillus subtilis) strains . Nauclea latifolia extracts were bacteriostatic to both Gram positive and Gram negative strains . No test strain was susceptible to the hot water extracts of Nauclea latifolia but five and seven strains, were respectively susceptible to similar extracts of Anthocliesta djalonensis and Uvaria afzalii . Of the seven column chromatographic fractions of the ethanolic extract of Uvaria afzalii, F(ua-1) exhibited a bactericidal activity restricted to the Gram negative Escherichia coli strains, which were not susceptible to the crude extract . Fractions, F(ua-2), F(ua-3) and F(ua-4), like the crude extract, were bactericidal against the Gram positive strains only . Thus, partial purification seems to broaden the spectrum of activity and generally improve the potency of Uvaria afzalii . These results apparently justify the use of the three plants in treatment of STD .

Acta Microbiol Pol, 2003, 52(4), 419 - 23
Adherence of Pseudomonas aeruginosa to human buccal epithelial cells; Wolska K et al.; The aim of this study was to evaluate adherence of 83 strains of Pseudomonas aeruginosa isolated from humans and different animals to trypsin-treated buccal cells . We have demonstrated that Pseudomonas aeruginosa attached to trypsin-treated buccal cells in far greater numbers than to cells from controls (normal buccal epithelial cells) . The mean number of bacteria adhering to trypsin-treated cells amounted 107.05 +/- 102.16 and to normal cells - 6.97 +/- 3.53 . We conclude that exposure of cells to proteolytic enzymes increases Pseudomonas aeruginosa binding to buccal cells.

Pediatr Pulmonol, 2004 May, 37(5), 427 - 32
Antibody response to Pseudomonas aeruginosa in cystic fibrosis patients: a marker of therapeutic success?--A 30-year cohort study of survival in Danish CF patients after onset of chronic P . aeruginosa lung infection; Johansen HK et al.; We studied the effects of increasingly intensive treatment regimens on anti-pseudomonal antibody response and survival in five successive cohorts of a total of 157 Danish cystic fibrosis patients after they had acquired chronic P . aeruginosa lung infection . The time periods were 1971-1975 (N = 21), 1976-1980 (N = 64), 1981-1986 (N = 27), 1987-1993 (N = 26), and 1994-2000 (N = 19) . During this 30-year period, we introduced elective 2-week courses of chemotherapy every third month in all chronically infected patients, early aggressive treatment with inhalation of colistin and oral ciprofloxacin for 3 months whenever P . aeruginosa was cultured in sputum from noncolonized patients, and inhalation of recombinant human dornase alfa . There was a significant correlation between the calendar year when chronic P . aeruginosa infection was acquired and the subsequent increase in the level of precipitins (P < 0.00001) . The median number of precipitins increased by 5 per year in the oldest calendar year cohort, and 1 per year in the youngest . The median age of onset of chronic P . aeruginosa increased from 9.3 years from 1981-1986 to 13.8 years from 1987-2000 . Survival after acquisition of chronic P . aeruginosa lung infection improved with time (P = 0.008) . Our study shows that CF patients who are treated intensively have lower antibody responses and longer survival after acquisition of chronic P . aeruginosa lung infection .

Pediatr Pulmonol, 2004 May, 37(5), 407 - 12
Disease severity in siblings with cystic fibrosis; Katz SL et al.; Since cross-infection occurs between cystic fibrosis (CF) siblings, we hypothesized that subsequent siblings may acquire respiratory pathogens at an earlier age and have a more severe course of pulmonary disease . We studied a retrospective cohort of 31 sibling pairs from the CF database at the Hospital for Sick Children . Kaplan-Meier curves and modified log-rank tests were used to test sibling differences in age of acquisition of Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), or any positive culture . Differences in disease severity outcomes were explored . Older siblings were more likely to have both SA and any CF pathogen first isolated from respiratory culture at an older age than younger siblings (P = 0.0050 and P = 0.0008, respectively, by modified log-rank tests) . However, more of the older siblings were positive on first culture at time of diagnosis, introducing an age-of-diagnosis bias . Hospitalization rates, courses of oral antibiotics, FEV(1) % predicted, and weight and height measurements were not better in the older children . No differences in clinical parameters were found between older and younger siblings . The apparent finding of younger age at first isolation of pathogens from respiratory cultures in younger siblings is likely because many older siblings were already infected with these organisms at time of diagnosis .

Pediatr Pulmonol, 2004 May, 37(5), 400 - 6
Pulmonary exacerbations in cystic fibrosis; Rabin HR et al.; The clinical characteristics most relevant to the decision to treat for a pulmonary exacerbation with antibiotics in cystic fibrosis patients were determined . Variables including age, increased cough frequency and sputum production, new crackles and wheezing, asthma, symptomatic sinusitis, hemoptysis, decreased lung function, weight loss, and new acquisition of Pseudomonas aeruginosa were collected in a large prospective multicenter database (Epidemiologic Study of Cystic Fibrosis) . During a 12-month baseline period, data from 11692 patients were compared with data collected during the subsequent 6-month study period . Because pulmonary function assessments were unavailable for patients <6 years of age, separate analyses were done for those <6 and >or=6 years of age . The outcome of interest was any antibiotic treatment in the 6-month study period reported as indicated for an exacerbation . Characteristics with the most discriminatory power were determined using stepwise multiple logistic regression . For patients <6 years of age, the strongest independent associations with treatment for a pulmonary exacerbation were new crackles, increased cough frequency, decline in weight, and increased sputum production . For those patients >or=6 years of age, the strongest independent associations were a relative decrease in percent predicted forced expired volume in 1 sec, increased cough frequency, new crackles, and hemoptysis . The presence of three or more of these key characteristics was strongly associated with the occurrence of a treated exacerbation . The reproducibility of the model over time was confirmed by application to a subsequent set of data . This model has potential for use as an outcome measure in clinical trials, and to assist in treatment decisions for individual patients .

Eye, 2004 Sep, 18(9), 935 - 7
Continuous wear silicone hydrogel contact lenses and microbial keratitis; Whiting MA et al.; Like other lens types, the new generation of silicone hydrogel contact lenses can be associated with a spectrum of ocular complications . Most tend to be very minor, but serious and sight-threatening complications can occur . We present four such cases with microbial keratitis following extended wear of these lenses . Cultures were positive for Pseudomonas aeruginosa in three cases and all three of these suffered lasting visual impairment . We describe our findings and discuss possible risk factors.

FEBS Lett, 2004 Apr 23, 564(1-2), 69 - 72
Characterisation of the PQQ cofactor radical in quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa by electron paramagnetic resonance spectroscopy; Kay CW et al.; The binding pocket of the pyrroloquinoline quinone (PQQ) cofactor in quinoprotein alcohol dehydrogenases contains a characteristic disulphide ring formed by two adjacent cysteine residues . To analyse the function of this unusual structural motif we have investigated the wild-type and a double cysteine:alanine mutant of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa by electron paramagnetic resonance (EPR) spectroscopy . Thus, we have obtained the principal values for the full rhombic g-tensor of the PQQ semiquinone radical by high-field (94 GHz) EPR necessary for a discrimination of radical species in dehydrogenases containing PQQ together with other redox-active cofactors . Our results show that the characteristic disulphide ring is no prerequisite for the formation of the functionally important semiquinone form of PQQ.

J Cataract Refract Surg, 2004 Apr, 30(4), 884 - 91
Prevention of experimental diffuse lamellar keratitis using a novel platelet-activating factor receptor antagonist; Esquenazi S et al.; PURPOSE: To determine whether a novel platelet-activating factor (PAF) antagonist prevents experimentally induced diffuse lamellar keratitis (DLK) after laser in situ keratomileusis (LASIK) . SETTINGS: Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Science Center, New Orleans, Louisiana, USA . METHODS: Twenty eyes of 10 New Zealand albino rabbits were used . The left eyes were treated with a peribulbar injection of 0.5 mL of PAF receptor antagonist LAU 0901 (2,4,6-trimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid ester) dissolved in 20 hydroxypropyl B cyclodextrin (30 microg /mL) . Two rabbits were treated with a peribulbar injection of 0.5 mL of vehicle (cyclodextrin) alone and served as controls . A corneal flap was cut in all eyes, and the interface was exposed to Pseudomonas aeruginosa endotoxin . The left eyes were additionally treated with 1 drop of LAU 0901 4 times a day . Rabbits were killed on postoperative days 1, 2, 3, 5, and 8 . The eyes were enucleated and processed for histopathology and immunohistochemical examination . RESULTS: Corneas not treated with LAU 0901 and controls showed a severe inflammatory response in the flap margin and stromal interface, characterized by loss of keratocytes, activation of adjacent keratocytes and transformation to myofibroblasts, infiltration of polymorphonuclear leukocytes and monocytes, and presence of epithelial cells with necrosis and melting of adjacent stroma . Corneas of rabbits treated with LAU 0901 showed minimal loss of keratocytes and myofibroblast transformation, minimal inflammatory cell infiltration, and minimal presence of epithelial cells in the interface . CONCLUSION: Induction of DLK was blocked by a PAF receptor antagonist in rabbit eyes . The histopathological evaluation and immunohistochemical studies showed that treatment with LAU 0901 blocked keratocyte apoptosis, transformation of fibroblasts to myofibroblasts and migration to the wound site, and chemotaxis of inflammatory cells, inhibiting the inflammatory response and promoting adequate healing of the flap interface and adjacent stroma.

J Bacteriol, 2004 May, 186(9), 2523 - 31
Structural and genetic characterization of glycosylation of type a flagellin in Pseudomonas aeruginosa; Schirm M et al.; Type a flagellins from two strains of Pseudomonas aeruginosa, strains PAK and JJ692, were found to be glycosylated with unique glycan structures . In both cases, two sites of O-linked glycosylation were identified on each monomer, and these sites were localized to the central, surface-exposed domain of the monomer in the assembled filament . The PAK flagellin was modified with a heterogeneous glycan comprising up to 11 monosaccharide units that were O linked through a rhamnose residue to the protein backbone . The flagellin of JJ692 was less complex and had a single rhamnose substitution at each site . The role of the glycosylation island gene cluster in the production of each of these glycosyl moieties was investigated . These studies revealed that the orfA and orfN genes were required for attachment of the heterologous glycan and the proximal rhamnose residue, respectively.

J Spinal Disord, 2004 Apr, 17(2), 112 - 114
Histopathologic Effects of Discitis on Neural Tissues : An Experimental Study; Yucesoy K et al.; To determine the cause of neurologic symptoms and signs seen in discitis, the neural histopathologic effects of discitis were investigated in an experimental study carried out on rats . Groups of seven rats each had their intervertebral discs inoculated with either Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, or a control solution . Histopathologic examinations of the spinal cord and nerve roots were performed after 3 weeks . On histopathologic examination, vacuolar myelopathy in the spinal cord and vacuolar neuropathy within the nerve roots near the junction with the spinal cord were found . The severity and form of vacuolar myelopathy varied according to the bacteria used for inoculation . The myelopathy and neuropathy seen in this rat model of bacterial discitis might be the result of an immunologic mechanism and could be responsible for the neurologic signs and symptoms of discitis in patients.

Shock, 2004 May, 21(5), 458 - 65
Selective inducible nitric oxide synthase inhibition during long-term hyperdynamic porcine bacteremia; Matejovic M et al.; We have recently demonstrated that selective inducible nitric oxide (NO) synthase (iNOS) inhibition with 1400W attenuated the hemodynamic and metabolic alterations affiliated with hyperdynamic porcine endotoxemia . In contrast to endotoxemia, limited evidence is available to document a relationship between NO and organ dysfunction in large animal bacteremic models . Therefore, using the same experimental setup, we investigated the role of selective iNOS blockade in porcine bacteremia induced and maintained for 24 h with a continuous infusion of live Pseudomonas aeruginosa . After 12 h of sepsis, animals received either vehicle (Control, n = 8) or continuous infusion of selective iNOS inhibitor, L-N6-(1-iminoethyl)-lysine (L-NIL; n = 8) . Measurements were performed before, and 12, 18, and 24 h after P . aeruginosa infusion . L-NIL inhibited sepsis-induced increase in plasma nitrate/nitrite concentrations and prevented hypotension without affecting cardiac output . Despite comparable hepatosplanchnic macrocirculation, L-NIL blunted the progressive deterioration in ileal mucosal microcirculation and prevented mucosal acidosis . L-NIL largely attenuated mesenteric and hepatic venous acidosis, significantly improved P . aeruginosa-induced impairment of hepatosplanchnic redox state, and mitigated the decline in liver lactate clearance . Furthermore, the administration of L-NIL reduced the hepatocellular injury and prevented the development of renal dysfunction . Finally, treatment with L-NIL significantly attenuated the formation of 8-isoprostane concentrations, a direct marker of lipid peroxidation . Thus, selective iNOS inhibition with L-NIL prevented live bacteria from causing key features of metabolic derangements in porcine hyperdynamic sepsis . Underlying mechanisms probably include reduced oxidative stress with improved microcirculatory perfusion and restoration of cellular respiration.

Shock, 2004 May, 21(5), 444 - 51
Massive alveolar thrombin activation in Pseudomonas aeruginosa-induced acute lung injury; Kipnis E et al.; In acute lung injury (ALI), a coagulation/fibrinolysis imbalance leads to fibrin deposition, persistence of which contributes to fibrotic evolution . Our study evaluated the effects of early inhibition of coagulation in Pseudomonas aeruginosa (Pa)-induced ALI through the use of recombinant human antithrombin (rhAT) . The study was conducted in vivo on a murine model of Pa-induced ALI . Intravenous rhAT was administered simultaneously with intratracheal Pa . Four experimental groups were compared: CTR, intratracheal saline (0.5 mL/kg)/intravenous saline (1 mL); PNP, intratracheal Pa (0.5 mL/kg of 2 x 10(9) cfu)/intravenous saline; AT, intratracheal saline/intravenous rhAT (500 IU/kg); ATPNP, intratracheal Pa/intravenous rhAT . Epithelial and endothelial permeabilities were evaluated with radiolabeled albumin flux across the alveolar barrier (125I- and 131I-labeled albumin) . Thrombin-antithrombin (TAT) complexes levels were used as markers of coagulation activation in blood samples and in BAL fluid . Epithelial and endothelial protein permeability were increased in Pa-induced ALI versus control . Intravenous rhAT administration led to further permeability disorders . Administration of rhAT in Pa ALI led to a rise in TAT complexes in ATPNP blood serum and BAL fluids compared with the other groups . In Pa-induced ALI the administration intravenous rhAT leads to major histologic damage, alveolar capillary barrier injury, and permeability increase . Such effects of the inhibition of thrombin activation by rhAT lead to the hypothesis of a probable beneficial role of early coagulation activation in ALI as a factor limiting both the extent of injury and permeability disorders . Our study suggests that inhibition of this initial procoagulative imbalance is potentially dangerous.

Shock, 2004 May, 21(5), 415 - 25
Diminished bacterial clearance is associated with decreased IL-12 and interferon-gamma production but a sustained proinflammatory response in a murine model of postseptic immunosuppression; Murphey ED et al.; After a major illness or injury, immune status in critically ill patients may fluctuate between a marked proinflammatory response and an immunosuppressed state . Postinflammatory immunosuppression can result in increased susceptibility to infection . Alterations of cytokine production, such as suppression of IFNgamma and elevation of the anti-inflammatory cytokine IL-10, are believed to contribute to postinflammatory immunosuppression . We examined antimicrobial immunity in mice that had previously been subjected to a sublethal cecal ligation and puncture (CLP) as a model of major injury . Mice were challenged with Pseudomonas aeruginosa (5 x 10(7) CFU i.v.) on day 5 after CLP or sham surgery . Bacterial clearance in mice after CLP was impaired and associated with decreased production of IFNgamma and increased production of IL-10 in the early response to the Pseudomonas challenge . Pseudomonas-induced production of the IFNgamma-inducing factor IL-12 was also decreased in post-CLP mice . However, splenocytes from post-CLP mice remained responsive to exogenous stimulation with the IFNgamma-inducing cytokines IL-12, IL-15, and IL-18 as well as T-cell receptor activation . Furthermore, production of the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 were as high, or higher, in the post-CLP group compared with sham mice after P . aeruginosa challenge . Blockade of IL-10 did not reverse IL-12 and IFNgamma suppression in splenocytes from post-CLP mice . These studies show that suppressed bacterial clearance in post-CLP mice is associated with decreased production of IFNgamma and IL-12 and with increased production of IL-10 and proinflammatory cytokines.

Chemotherapy, 2004 Apr, 50(1), 31 - 4
Comparative activity of beta-lactam agents (carbapenem excepted) against Pseudomonas aeruginosa strains with CARB or OXA beta-lactamases; Bert F et al.; BACKGROUND: Ticarcillin resistance in Pseudomonas aeruginosa can be mediated by various beta-lactamases of the CARB and OXA groups . METHODS: We investigated the activity of seven beta-lactam agents and two beta-lactam-beta-lactamase inhibitor combinations against 216 P.aeruginosa strains with genotypically characterized beta-lactamases, including 137 CARB, 31 OXA-35, 25 OXA-10, 13 OXA-1 and 10 OXA-2 . MICs were determined by the agar dilution method . RESULTS: The activities of ticarcillin and piperacillin were more affected by CARB than by OXA enzymes . beta-Lactamase inhibitors were poorly effective against OXA-1, OXA-35 and OXA-10 . OXA-1 conferred resistance to cefepime and cefpirome but not to cefsulodin and aztreonam . Ceftazidime remained the most active agent against all groups of enzymes . Major differences in the susceptibility rates according to NCCLS and CASFM breakpoints were observed . CONCLUSIONS: Significant differences were found in the resistance profile associated with the various types of CARB and OXA beta-lactamases in P . aeruginosa .

Chemotherapy, 2004 Apr, 50(1), 27 - 30
Serum bactericidal activity of piperacillin/tazobactam against Staphylococcus aureus, piperacillin-susceptible and piperacillin-resistant Escherichia coli and Pseudomonas aeruginosa; Lemmen SW et al.; BACKGROUND: The serum bactericidal test measures the highest level of an antibiotic-containing serum dilution at which 99.9% of bacteria are killed . In this study the serum bactericidal activity of piperacillin/tazobactam was determined for bacteria often involved in severe infections . In earlier studies titres >/=1:8 in the serum bactericidal tests correlated well with clinical success in the treatment of endocarditis and osteomyelitis as well as bacterial eradication . METHODS: Blood samples of 6 healthy volunteers were taken before and 1 and 4 h after piperacillin/tazobactam (4.5 g) administration . Serum concentrations and serum bactericidal activity were determined for 10 strains each of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, both piperacillin-resistant and piperacillin-susceptible according to NCCLS guidelines . RESULTS: 100% of S . aureus and piperacillin-susceptible E . coli, 90% of piperacillin-resistant E . coli and 80% of P . aeruginosa were killed 1 h after drug administration . 4 h after drug administration serum bactericidal activity decreased to 60% for S . aureus, 90% for piperacillin-susceptible E . coli, 80% for piperacillin-resistant E . coli and 30% for P . aeruginosa . CONCLUSIONS: Excellent serum bactericidal activity of piperacillin/tazobactam was recorded 1 h after drug administration for S . aureus, E . coli and P . aeruginosa . After 4 h limited killing rates for P . aeruginosa could be detected, which supports the idea of a combination therapy .

Chemotherapy, 2004 Apr, 50(1), 22 - 6
A multidrug efflux pump inhibitor reduces fluoroquinolone resistance in Pseudomonas aeruginosa isolates; Coban AY et al.; In general, resistance to fluoroquinolones (FQs) in gram-negative bacteria is acquired either by mutations in DNA gyrase and topoisomerase IV or by active export of the agents via antibiotic efflux pumps . Reduced porin expression is also proposed to be another mechanism leading to resistance . In this study, interaction between levofloxacin, ofloxacin, and ciprofloxacin with MC-207,110 (multidrug efflux pump inhibitor) was investigated by a checkerboard assay using Pseudomonas aeruginosa . Levofloxacin, ofloxacin, and ciprofloxacin were tested at different concentrations (0.06-64 microg/ml) and MC-207,110 was tested at a concentration range of 4-128 microg/ml . In the presence of MC-207,110 (at 128, 64, 32, 16 microg/ml) resistance to FQs was inhibited significantly and MIC values were decreased, except at 8 and 4 microg/ml of MC-207,110 . When MC-207,110 was used, resistance of P . aeruginosa to FQs in vitro was inhibited significantly, suggesting that MC-207,110 may be useful for use in clinical treatment protocols to overcome FQs resistance .

Proc Natl Acad Sci U S A, 2004 Apr 27, 101(17), 6664 - 8 Epub 2004 Apr 14.
Pseudomonas aeruginosa regulates flagellin expression as part of a global response to airway fluid from cystic fibrosis patients; Wolfgang MC et al.; Cystic fibrosis (CF) patients are highly susceptible to chronic lung infections by the environmental bacterium Pseudomonas aeruginosa . The overproduction and accumulation of dehydrated viscous respiratory mucus and excessive inflammation represents a defining feature of CF and constitutes the major environment encountered by P . aeruginosa during chronic infections . We applied whole-genome microarray technology to investigate the ability of P . aeruginosa to respond to signals found in muco-purulent airway liquids collected from chronically infected CF patients . Particularly notable was the activation of the Rhl-dependent quorum-sensing (QS) network and repression of fliC, which encodes flagellin . Activation of the Rhl branch of the QS network supports the observation that QS molecules are produced in the chronically infected CF lung . The shut-off of flagellin synthesis in response to CF airway liquids was rapid and independent of QS and the known regulatory networks controlling the hierarchical expression of flagellar genes . As flagellin is highly immunogenic and subject to detection by host pattern recognition receptors, its repression may represent an adaptive response that allows P . aeruginosa to avoid detection by host defense mechanisms and phagocytosis during the chronic phase of CF lung infections.

Biotechnol Bioeng, 2004 May 5, 86(3), 365 - 73
Kinetics and mechanism of a reaction catalyzed by PST-01 protease from Pseudomonas aeruginosa PST-01; Bobe IM et al.; The initial rates of carboxybenzoyl-alanyl-l-leucyl-amide (Z-L-Ala-L-Leu-NH(2)) synthesis from carboxybenzoyl-L-alanine (Z-L-Ala) and L-leucineamide (L-Leu-NH(2)) and Z-L-Ala-L-Leu-NH(2) hydrolysis in a homogeneous dimethyl sulfoxide-aqueous buffer solution {1:1 (v/v)} system catalyzed by PST-01 protease from Pseudomonas aeruginosa were measured under a wide range of Z-L-Ala, L-Leu-NH(2) and Z-L-Ala-L-Leu-NH(2) concentrations . The initial rates of the synthetic reaction, in which Z-L-Ala-L-Leu-NH(2) was produced from Z-L-Ala and L-Leu-NH(2), were inhibited by the substrates . Furthermore, the initial rates of the synthetic reaction were not inhibited by the product Z-L-Ala-L-Leu-NH(2), and those of the hydrolytic reaction were inhibited by Z-L-Ala and L-Leu-NH(2) . All the initial rate data of the synthetic and hydrolytic reactions were well correlated with the rate equation derived based on the proposed reaction scheme .

Int J Pharm, 2004 May 4, 275(1-2), 171 - 87
Factorial design, physicochemical characterisation and activity of ciprofloxacin-PLGA nanoparticles; Dillen K et al.; Poly(lactide-co-glycolide) nanoparticles incorporating ciprofloxacin HCl were prepared by means of a W/O/W emulsification solvent evaporation method . The stabiliser selected was poly(vinylalcohol) . A 2(4) full factorial design based on four independent variables was used to plan the experiments and the variable parameters were the number of homogenisation cycles, addition of boric acid to the inner water phase containing the drug, ciprofloxacin concentration in the inner water phase and oil:outer water phase ratio . The effects of these parameters on the particle size, zeta potential, drug loading efficiency and drug release were investigated . Also the effect of gamma irradiation on the particle size and drug release was evaluated and DSC and XRD analyses of the compounds and the nanoparticles were performed . The activity on two series of microorganisms, Pseudomonas aeruginosa and Staphylococcus aureus, was examined.

Int J Antimicrob Agents, 2004 Apr, 23(4), 401 - 4
Frequency of Gram-negative bacterial pathogens in bloodstream infections and their resistance to antibiotics in the Czech Republic; Cermak P et al.; A study performed at 12 hospitals in the Czech Republic in 2001 evaluated the Gram-negative bacterial pathogens most frequently associated with bloodstream infections and their susceptibility to a selection of antimicrobial agents . Of 831 Gram-negative strains, the most frequently isolated organisms were Escherichia coli (32%), Klebsiella pneumoniae (24%) and Pseudomonas aeruginosa (10%) . E . coli isolates were relatively susceptible to the antibiotics tested, whereas K . pneumoniae were relatively resistant to all agents except meropenem, and P . aeruginosa to all agents except gentamicin and amikacin . Other agents showed variable rates of resistance to penicillins, third-generation cephalosporins, aminoglycosides and ciprofloxacin.

J Appl Microbiol, 2004, 96(5), 1124 - 32
A rapid method for the evaluation of both extrinsic and intrinsic contamination and resulting spoilage of water-in-oil emulsions; O'May GA et al.; AIMS: To develop a method for studying the microbial spoilage of water-in-oil emulsions and to use this to investigate (i) the intrinsic stability of water-in-oil formulations and (ii) Pseudomonas aeruginosa SP1-induced spoilage of a proprietary emulsion . METHODS AND RESULTS: Aliquots of test emulsion were placed into wells of a microtitre plate and the opacity (492 nm) monitored at 120-min intervals over several hours . Cracking of the emulsion was associated with marked reductions in opacity . Rate and extent of change in O.D . could be used as indicators of spoilage . Spoilage of a laboratory emulsion formulation was investigated where microorganisms with demonstrated spoilage potential were incorporated either into the water phase prior to emulsification or where the proportion of contaminated water droplets was varied by dilution of contaminated emulsion with a sterile formulation . Results suggested that the route of introduction was a critical determinant of the probability of gross spoilage . Ps . aeruginosa SP1-induced spoilage of a proprietary formulation was found to be independent of growth in the formulation; rather it was attributed to the presence of a heat-labile extracellular spoilage-factor that was protease labile and possessed both lipase and polysorbate hydrolytic activity . Such spoilage potential was unique to one Ps . aeruginosa culture filtrate amongst five pseudomonads tested . SIGNIFICANCE AND IMPACT OF THE STUDY: The method is both rapid and reproducible, enables evaluation of the effects of route of contamination upon emulsion spoilage and has potential application in formulation development for cosmetic, pharmaceutical and food products.

Protein Sci, 2004 May, 13(5), 1260 - 5 Epub 2004 Apr 09.
Structure of fosfomycin resistance protein FosA from transposon Tn2921; Pakhomova S et al.; The crystal structure of fosfomycin resistance protein FosA from transposon Tn2921 has been established at a resolution of 2.5 A . The protein crystallized without bound Mn(II) and K+, ions crucial for efficient catalysis, providing a structure of the apo enzyme . The protein maintains the three-dimensional domain-swapped arrangement of the paired betaalphabetabetabeta-motifs observed in the genomically encoded homologous enzyme from Pseudomonas aeruginosa (PA1129) . The basic architecture of the active site is also maintained, despite the absence of the catalytically essential Mn(II) . However, the absence of K+, which has been shown to enhance enzymatic activity, appears to contribute to conformational heterogeneity in the K(+)-binding loops.

Mikrobiologiia, 2004 Jan-Feb, 73(1), 45 - 50
{The requirements of Pseudomonas aeruginosa dissociants for carbon, nitrogen, and phosphorus}; Fursova PV et al.; Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation . The requirements of the R, S, and M dissociants of Pseudomonas aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments . For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants . The nutritional requirements of the dissociants are discussed in relation to our earlier data.

Mikrobiologiia, 2004 Jan-Feb, 73(1), 37 - 44
{Effect of major nutrient elements on the growth and population homogeneity of the R, S and M dissociants of Pseudomonas aeruginosa and the glucose oxidation and fermentation pathways}; Mil'ko ES et al.; The population homogeneity of the stationary-phase monocultures of Pseudomonas aeruginosa dissociants was studied as a function of the initial content of major nutrient elements (C, N, and P) in the cultivation medium . The monocultures of the dissociants remained homogeneous during cultivation if the initial concentrations of the major nutrient elements were either sufficiently high or, conversely, very low, but became heterogeneous during cultivation in unbalanced (with respect to the major nutrient elements) media . At the initial concentration of nitrate in the medium equal to 0.07% or phosphate equal to 0.004-0.014%, the initially homogeneous population of R dissociant cultivated to the stationary growth phase turned out to contain 30-40% of S-type cells, whereas the initially homogeneous population of S dissociant was found to contain 50-80% of M-type cells . The population of M dissociant remained homogeneous throughout the cultivation period . R dissociant grew better at sufficiently high concentrations of glucose, nitrate, and phosphate in the medium, whereas M dissociant grew better when the initial concentrations of these nutrients were low . During the cultivation of R dissociant, the pH of the medium changed insignificantly, and the C/P ratio (the ratio of the carbon and phosphorus consumed during growth) was minimal (among the three dissociants), indicating that the R dissociant accomplishes the oxidative pathway of glucose metabolism . During the cultivation of the M dissociant, the pH of the medium dropped to 3.4-3.9, and the C/P ratio was maximal, indicating that this dissociant accomplishes the fermentative pathway of glucose metabolism . During the cultivation of the S dissociant, the pH of the medium and the C/P ratio exhibited variations, indicating that this dissociant triggers its pathways of glucose metabolism.

Microbiology, 2004 Apr, 150(Pt 4), 967 - 78
Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates; Viana-Niero C et al.; The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes . Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC) . The fourth gene, plcD, is located in a different region . This study investigates variations in plcABC and plcD genes in clinical isolates of M . tuberculosis, Mycobacterium africanum and 'Mycobacterium canettii' . Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR . Seven M . tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD . In 19 of 25 M . tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci . Partial plcD deletion was observed in one M . africanum isolate . In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation . A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M . tuberculosis isolates . Five M . tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes . Phospholipase C is a well-known bacterial virulence factor . The precise role of phospholipase C in the pathogenicity of M . tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

Microbiology, 2004 Apr, 150(Pt 4), 831 - 41
Global regulation of quorum sensing and virulence by VqsR in Pseudomonas aeruginosa; Juhas M et al.; Pathogenesis of Pseudomonas aeruginosa is controlled to a major extent by the two quorum-sensing systems las and rhl . The previously uncharacterized gene PA2591 was identified as a major virulence regulator, vqsR, in the quorum-sensing hierarchy . vqsR is a member of the LuxR family and possesses a las box in its upstream region . Transposon inactivation of vqsR abrogated the production of N-acylhomoserine lactones and the secretion of exoproducts and diminished bacterial virulence for Caenorhabditis elegans . Cytotoxicity towards macrophages was not affected . vqsR mRNA was expressed more strongly in the presence of human serum and oxidative stress than under standard growth conditions . High-density oligonucleotide microarrays were used to compare the global expression profile of a wild-type strain and a vqsR mutant . One-hundred-and-fifty-one and 113 genes were significantly differentially expressed in the presence of H(2)O(2) and human serum, respectively . The disruption of vqsR repressed the expression of genes that are known to be promoted by quorum sensing and activated the expression of genes that are known to be repressed by quorum sensing . Moreover, the vqsR mutant harboured less mRNA transcript for the production of siderophores and membrane-bound elements of antibiotic resistance . The protein encoded by PA2591 regulates several traits of pathogenicity; hence, the name vqsR ('virulence and quorum-sensing regulator') was assigned to PA2591.

Microbiology, 2004 Apr, 150(Pt 4), 795 - 803
Genetic characterization of pcpS, encoding the multifunctional phosphopantetheinyl transferase of Pseudomonas aeruginosa; Barekzi N et al.; Fatty acid synthases (primary metabolism), non-ribosomal peptide synthases and polyketide synthases (secondary metabolism) contain phosphopantetheinyl (Ppant)-dependent carrier proteins that must be made functionally active by transfer of the 4'-Ppant moiety from coenzyme A . These reactions are usually catalysed by dedicated Ppant transferases . Although rich in Ppant-dependent carrier proteins, it was previously shown that Pseudomonas aeruginosa possesses only one Ppant transferase, encoded by pcpS, which functions in both primary and secondary metabolism . Consistent with this notion are our findings that pcpS can genetically complement mutations in the Escherichia coli acpS and entD genes, encoding the apo-acyl carrier protein (ACP) synthase of fatty acid synthesis and a Ppant transferase of enterobactin synthesis, respectively . It also complements a Bacillus subtilis sfp mutation affecting a gene encoding a Ppant transferase essential for surfactin synthesis . A pcpS insertion mutant could only be constructed in a strain carrying the E . coli acpS gene on a chromosomally integrated element in trans, implying that the in vitro essentiality of pcpS is due to its requirement for activation of apo-ACP of fatty acid synthesis . The conditional pcpS mutant is non-fluorescent, does not produce pyoverdine and pyochelin, and does not grow in the presence of iron chelators . The data presented here for the first time confirm that PcpS plays an essential role in both fatty acid and siderophore metabolism.

J Antimicrob Chemother, 2004 May, 53(5), 693 - 5 Epub 2004 Apr 08.
Whither triclosan?
Russell AD.
Triclosan has activity against many, but not all, types of Gram-positive and Gram-negative bacteria . It is bacteriostatic at low concentrations, but higher concentrations are bactericidal . Pseudomonas aeruginosa is highly resistant, whereas methicillin-resistant Staphylococcus aureus strains are inhibited over a range of approximately 0.1-2 mg/L . Triclosan shows significant activity against some mycobacteria, but is not sporicidal . Its growth-inhibitory properties result from an inhibition of enoyl reductase, FabI . Membrane-destabilizing effects are likely to be responsible for bacterial inactivation by higher concentrations . Resistance can arise from mutations in, and/or overproduction of, FabI, impermeability or efflux . Whilst triclosan resistance in laboratory experiments may be associated with changes in antibiotic susceptibility, comprehensive environmental surveys have not demonstrated any association between triclosan usage and antibiotic resistance . Triclosan has several important uses, and the future aim must be to retain these applications whilst eliminating the more frivolous and unnecessary ones.

J Indian Med Assoc, 2003 Aug, 101(8), 490 - 2
Nosocomial ocular infection--a prospective study; Das A et al.; Nosocomial infection of the eye is an uncommon complication, acquired during the course of hospital management . It may prolong the disease process or even destroy the eye . The overall incidence varies considerably by hospital services . To ascertain the various types of ocular infections and its responsible pathogens, a laboratory-based, nosocomial ocular infection control study was performed in a large referral hospital during a period of January 1997 to June 1999 . The study revealed 29 cases (0.08%) of culture proven ocular infections, out of 35,758 total admission during the period of one calendar year . Fifty-one eyes of 29 cases (22 bilateral) had nosocomial infection . Staphylococcus aureus (9), Staphylococcus epidermidis (8) and Pseudomonas aeruginosa (5), were the most frequent bacteria . Laboratory investigations helped in initiation and modification of specific antimicrobial therapy and also prognosis . Proper surveillance with the help of laboratory investigations has effective role in the management of nosocomial ocular infection.

Mol Diagn, 2003, 7(3-4), 195 - 200
PCR-based detection of a cystic fibrosis epidemic strain of Pseudomonas Aeruginosa; Panagea S et al.; BACKGROUND: The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is widespread among patients with cystic fibrosis (CF) in specialist centers around Liverpool and elsewhere in the UK . This study evaluates a new diagnostic PCR assay based on a unique DNA sequence (PS21) of LES, for its identification of colonies directly from sputum . METHODS: One hundred and fifty-eight sputum samples from 92 patients were cultured and P . aeruginosa isolates were typed by PS21 PCR and pulsed-field gel electrophoresis (PFGE) . Subsequently, PS21 PCR was performed directly on sputum and the results were compared with culture, PFGE, and PS21 PCR typing . RESULTS: Eighty patients were colonized with P . aeruginosa, 63 by LES (79%) . There was 100% concordance between PS21 PCR on colonies and PFGE typing . The sensitivity and specificity of PS21 PCR directly on sputum was 98.2% and 93.6%, respectively . CONCLUSIONS: This study shows that PS21 PCR can be used for simple and rapid screening of LES colonization in CF patients.

Appl Environ Microbiol, 2004 Apr, 70(4), 2105 - 9
Use of whole cells of Pseudomonas aeruginosa for synthesis of the antioxidant hydroxytyrosol via conversion of tyrosol; Allouche N et al.; For the first time, a soil bacterium, designated Pseudomonas aeruginosa, was isolated based on its ability to grow on tyrosol as a sole source of carbon and energy . During growth on tyrosol, this strain was capable of promoting the formation of a significant amount of hydroxytyrosol and trace quantities of parahydroxyphenyl acetic acid and 3,4-dihydroxyphenyl acetic acid . The products were confirmed by high-performance liquid chromatography and gas chromatography-mass spectrometry analyses . Using an optimized tyrosol concentration of 2 g liter(-1), the maximal hydroxytyrosol yield (80%) was achieved after a 7-h reaction in a growth experiment . To enhance the formation of hydroxytyrosol and prevent its degradation, a resting-cell method using P . aeruginosa was performed . The growth state of the culture utilized for biomass production, the carbon source on which the biomass was grown, the concentration of the biomass, and the amount of tyrosol that was treated were optimized . The optimal yield of hydroxytyrosol (96%) was obtained after a 7-h reaction using 4 g of tyrosol liter(-1) and 5 g of cells liter(-1) pregrown on tyrosol and harvested at the end of the exponential phase . This proposed procedure is an alternative approach to obtain hydroxytyrosol in an environmentally friendly way . In addition, the reaction is easy to perform and can be adapted to a bioreactor for industrial purposes.

Philos Trans R Soc Lond B Biol Sci, 2004 Jan 29, 359(1441), 129 - 40
Hypervariability, suppressed recombination and the genetics of individuality; Olson MV et al.; We define 'genetic individuality' as intraspecies variation that has substantial heritability and involves traits that are sufficiently common that they can be observed in any modest-sized sampling of individuals . We propose that genetic individuality is largely shaped by the combinatory shuffling of a modest number of genes, each of which exists as a family of functionally and structurally diverged alleles . Unequivocal examples of such allele families are found at the O-antigen-biosynthetic locus in Pseudomonas aeruginosa and the human leucocyte antigen locus in humans . We examine characteristic features of these allele families and explore the possibility that genetic loci with similar characteristics can be recognized in a whole-genome scan of human genetic variation.

Oncogene, 2004 Jun 17, 23(28), 4894 - 902
Bacterial N-acylhomoserine lactone-induced apoptosis in breast carcinoma cells correlated with down-modulation of STAT3; Li L et al.; Cell growth is promoted by mitogens and survival factors, which activate intracellular signalling pathways to control cell cycle progression and cellular integrity . Proliferation signals are transmitted through Ras and Rho family small G-proteins coupled to mitogen-activated protein kinase (MAPK) cascades, while survival signals are propagated by lipid-dependent kinases such as phosphatidylinositide 3-kinases (PI3Ks) and protein kinase B (Akt/PKB) . Recently, signal transducer and activator of transcription (STAT) proteins were identified as positive regulators of proliferation in a variety of cell types . Persistent activation of these pathways is associated with tumour cell growth, whereas their inhibition can halt proliferation and precipitate apoptotic cell death . The human pathogen Pseudomonas aeruginosa uses quorum-sensing signal molecules (QSSMs) to regulate virulence gene expression . QSSMs also suppress host immune responses although the mechanism of suppression is unknown . Here, we demonstrate that the QSSM N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) from P . aeruginosa blocks proliferation and induces apoptosis in human BC cell lines . Analyses of signalling events reveal that OdDHL has little or no effect on MAPK cascades, partially inhibits the Akt/PKB pathway and ablates STAT3 activity . Pharmacological inhibition of each pathway independently indicates that STAT3 activity is critical for BC cell proliferation and survival, while a constitutively active STAT3 confers resistance to OdDHL . These results support the notion of OdDHL as a bioactive molecule in eukaryotic systems and a paradigm for a novel class of antiproliferative compounds .

J Microbiol Methods, 2004 May, 57(2), 163 - 73
Evaluation of disinfective potential of reactivated free chlorine in pooled tap water by electrolysis; Nakajima N et al.; Tap water is one of the causative factors of hospital infections . We examined the disinfective potential of electrolysis and mechanism of disinfection, and clarified the disinfective effect of electrolysis on tap water contaminated with bacteria, and discussed its clinical applications . Tap waters artificially contaminated with Pseudomonas aeruginosa, Escherichia coli, Legionella pneumophila, and Staphylococcus aureus could be sterilized by electrolysis at 20-30 mA for 5 min . A high-density suspension (10(6) CFU/ml) of a spore forming bacterium, Bacillus subtilis was not completely sterilized by electrolysis at 50 mA up to 30 min, but a low-density suspension (10(5) CFU/ml) was totally sterilized by electrolysis at 50 mA for 5 min . Electrolyzed P . aeruginosa changed morphologically, that is, there was bleb formation on the cell wall and irregular aggregation of cytoplasmic small granules . Moreover, cytoplasmic enzyme, nitrate reductase, was inactivated by the electrolysis . On the other hand, genomic DNA of the electrolyzed bacteria was not degenerated, therefore, their DNA polymerase activity was not completely inactivated . Consequently, the major agent in electrolysis for bactericidal action was considered to be free chlorine, and the possible bactericidal mechanism was by destruction of bacterial membranes, followed by the aggregation of peripheral cytoplasmic proteins . Electrolysis of tap water for both disinfecting contaminating bacteria and increasing the disinfectant capacity was considered effective with some limitations, particularly against high-density contamination by spore-forming bacteria . In clinical settings, electrolysis of tap water is considered effective to disinfect water for hand washing in operation theatres, and bathing water for immunocompromised hosts.

Tidsskr Nor Laegeforen, 2004 Apr 1, 124(7), 933 - 5
{Tattooing dyes and pigments contaminated with bacteria}; Charnock C; BACKGROUND: Obtaining a tattoo has become increasingly popular in recent years . However, the degree to which tattooing represents a health risk in the form of infections is still a matter of debate . The aim of the present study was to investigate the microbial content of tattooing dyes and pigments (colourants) . MATERIAL AND METHODS: Microbes present in colourants used for tattooing were counted and identified using standard techniques and rapid identification systems . RESULTS: Seven of in total twelve colourants contributed by five studios contained bacteria . No fungi were found . Three solutions contained more than 10 8 bacteria/ml, chiefly aerobic, Gram-negative rods including Pseudomonas aeruginosa . No primary pathogens were found, however, the high colony counts found for some samples may represent infectious doses of a wide range of bacteria . INTERPRETATION: Although required by current regulations to be sterile, a number of colourants were highly contaminated with bacteria . The detection of bacteria in colourants necessitates a review of the studios' internal control procedures . In addition, the appropriate monitoring body should initiate testing of colourants in order to ascertain if these are contaminated at the time of purchase, or become so during use.

J Bacteriol, 2004 Apr, 186(8), 2281 - 7
Pseudomonas aeruginosa autoinducer enters and functions in mammalian cells; Williams SC et al.; Quorum sensing (QS) is a cell density-dependent signaling mechanism used by many bacteria to control gene expression . Several recent reports indicate that the signaling molecules (autoinducers) that mediate QS in Pseudomonas aeruginosa may also modulate gene expression in host cells; however, the mechanisms are largely unknown . Here we show that two P . aeruginosa autoinducers, N-3-oxododecanoyl-homoserine lactone and N-butyryl-homoserine lactone, can both enter eukaryotic cells and activate artificial chimeric transcription factors based on their cognate transcriptional activators, LasR and RhlR, respectively . The autoinducers promoted nuclear localization of chimeric proteins containing the full LasR or RhlR coding region, and the LasR-based proteins were capable of activating transcription of a LasR-dependent luciferase gene . Responsiveness to autoinducer required the N-terminal autoinducer-binding domains of LasR and RhlR . Truncated proteins consisting of only the C-terminal helix-turn-helix DNA-binding domains of both proteins attached to a nuclear localization signal efficiently translocated to the nucleus in the absence of autoinducer, and truncated LasR-based proteins functioned as constitutively active transcription factors . Chimeric LasR proteins were only activated by their cognate autoinducer ligand and not by N-butyryl-L-homoserine lactone . These data provide evidence that autoinducer molecules from human pathogens can enter mammalian cells and suggest that autoinducers may influence gene expression in host cells by interacting with and activating as-yet-unidentified endogenous proteins.

Zhonghua Shao Shang Za Zhi, 2004 Feb, 20(1), 6 - 9
{Investigation on the drug resistance of Pseudomonas aeruginosa in our burn ward in the past 11 years}; Dou Y et al.; OBJECTIVE: To analyze the use of antibiotics and the drug resistance of Pseudomonas aeruginosa in the burn ward of our hospital in the past 11 years, so as to optimize the use of antibiotics in the future . METHODS: Bacterial epidemiology during 1991-2001 in our burn ward was investigated . The change of the drug resistance of Pseudomonas aeruginosa was observed by defined daily dose (DDD) of antibiotics in adult patients and by the ranking of antibiotic administration days . RESULTS: (1) Staphylococcus aureus (10.53%-34.40%) and Pseudomonas aeruginosa (75.66%-11.47%) were dominant in our burn ward . (2) Predominant antibiotics used included Penicillin, Amikacin, Vancomycin, Imipenem and Ceftazidime . (3) There was increasing drug resistance of Pseudomonas aeruginosa to the following antibiotics ranking in following order: Piperacillin (41.57%-100.00%), Imipenem (36.36%-98.46%), Ceftazidime (23.46%-97.85%), Amikacin (13.16%-100.00%) and ciprofloxacin (6.90%-100.00%) . CONCLUSION: There was increasing drug resistance of Pseudomonas aeruginosa to all antibiotics, which might be related to antibiotic abuse.

Lett Appl Microbiol, 2004, 38(5), 360 - 5
Methods for assaying cyanide in bacterial culture supernatant; Zlosnik JE et al.; AIMS: To find an easy, rapid and direct method for the quantitation of cyanide in a moderate number of bacterial culture supernatants . METHODS AND RESULTS: Culture supernatant from stationary phase cultures of Pseudomonas aeruginosa, grown in LB media, were analysed for cyanide content using the Merckoquant and Spectroquant cyanide detection kits as well as a cyanide ion-selective electrode (ISE) and a cyanide micro-ISE . The Merckoquant kit, designed for detection of low quantities of cyanide in water systems, proved not to be sufficiently reliable, providing poor comparison with previous assessments of cyanide levels in Ps . aeruginosa . The Spectroquant kit, and the two ISEs all provided very similar results, in agreement with previous data; however, it was the ISEs that fulfilled all the criteria for a rapid, direct test in a moderate number of samples . CONCLUSIONS: Cyanide ISEs can be used for easy assessment of the cyanide quantity in cultures grown in LB medium . Significance and Impact of the Study: The use of a cyanide ISE allows for an easy, direct and reproducible method for assaying cyanide in bacterial culture supernatant, which is of significant advantage over the currently accepted methods . This is especially important in an era of high-output genomic studies for assessing the phenotypic significance of data relating to the cyanide synthetic genes.

J Biol Chem, 2004 Jun 11, 279(24), 25830 - 7 Epub 2004 Mar 30.
The structure of the organic hydroperoxide resistance protein from Deinococcus radiodurans . Do conformational changes facilitate recycling of the redox disulfide?
Meunier-Jamin C, Kapp U, Leonard GA, McSweeney S.
The three-dimensional structure of the organic hydroperoxide resistance protein (OHRP) from Deinococcus radiodurans as determined using single crystal xray diffraction techniques is reported . Comparison of the structure with that obtained for OHRP from Pseudomonas aeruginosa reveals that the polypeptide chain of OHRPs can adopt two significantly different conformations ("in" and "out") in the region of the active site disulfide moiety . It is postulated that the closed configuration is consistent with efficient catalysis of the reduction of organic hydroperoxides, whereas the open form is required for enzyme recycling . Comparison of the structures of OHRP and that of the osmotically induced protein C (OsmC) from Mycoplasma pneumoniae shows that OHRPs and OsmCs are structurally homologous, perhaps indicating related functions for the two families of proteins.

Antimicrob Agents Chemother, 2004 Apr, 48(4), 1406 - 9
Molecular analysis of metallo-beta-lactamase gene bla(SPM-1)-surrounding sequences from disseminated Pseudomonas aeruginosa isolates in Recife, Brazil; Poirel L et al.; The spread of clonally related carbapenem-resistant Pseudomonas aeruginosa producing the metallo-beta-lactamase SPM-1 was found in Recife, Brazil . Upstream of bla(SPM-1), a novel common region (CR4) was identified, comprising an open reading frame, orf495, whose product shares significant identity with putative recombinases, such as Orf513 . CR4 may be responsible for mobilization and expression of bla(SPM-1).

Antimicrob Agents Chemother, 2004 Apr, 48(4), 1320 - 8
Enhancement of the mexAB-oprM efflux pump expression by a quorum-sensing autoinducer and its cancellation by a regulator, MexT, of the mexEF-oprN efflux pump operon in Pseudomonas aeruginosa; Maseda H et al.; nfxC-type cells of Pseudomonas aeruginosa that produce the MexEF-OprN efflux pump exhibit resistance to fluoroquinolones and chloramphenicol and hypersusceptibility to most classical beta-lactam antibiotics . We investigated the molecular mechanism of how the nfxC mutation causes beta-lactam hypersusceptibility . The MexAB-OprM extrusion pump transports and confers resistance to beta-lactam antibiotics . Interestingly, expression of the mexAB-oprM operon reached the highest level during the mid-stationary growth phase in both wild-type and nfxC-type mutant strains, suggesting that expression of the mexAB-oprM operon may be controlled by cell density-dependent regulation such as quorum sensing . This assumption was verified by demonstrating that exogenous addition of the quorum-sensing autoinducer N-butyryl-L-homoserine lactone (C4-HSL) enhanced the expression of MexAB-OprM, whereas N-(3-oxododecanoyl)-L-homoserine lactone had only a slight effect . Furthermore, this C4-HSL-mediated enhancement of mexAB-oprM expression was repressed by MexT, a positive regulator of the mexEF-oprN operon . It was concluded that beta-lactam hypersusceptibility in nfxC-type mutant cells is caused by MexT-mediated cancellation of C4-HSL-mediated enhancement of MexAB-OprM expression.

Biosens Bioelectron, 2004 May 15, 19(10), 1237 - 44
Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa; Ohfuji K et al.; Pyocyanin is the blue phenazine pigment produced by Pseudomonas aeruginosa . Pyocyanin production using immobilized cells was investigated . The maximum production of pyocyanin was obtained using cells immobilized in kappa-carrageenan . Moreover, 0.01% PO4(3-), 0.2% Mg(2+), 0.001% Fe(2+), 1% glycerine, 0.8% leucine and 0.8% dl-alanine were also essential for pyocyanin production . Pyocyanin was purified by chloroform extraction and silica gel column chromatography . An amperometric biosensor system using a screen-printed electrode and pyocyanin as mediator were also developed for a more accurate determination of glucose concentration . Pyocyanin, which exists in the oxidated form, was reduced by the reaction between glucose oxidase and glucose . The reduced form was then converted back to the oxidized form by an oxidative reaction on the electrode . There was a linear relation ship between sensor output currents and glucose concentrations ranging from 1 to 20mM under the following conditions: -200 mV of the applied potential, pH 5.0, and 10 U of the immobilized enzyme . The coefficient of variation was below 3% (n = 5) for the glucose sensor.

Can Respir J, 2004 Mar, 11(2), 151 - 5
Long term azithromycin therapy in cystic fibrosis patients: a study on drug levels and sputum properties; Baumann U et al.; BACKGROUND: Following reports on the treatment of diffuse panbronchiolitis (DPB), recent studies demonstrate that long term therapy with azithromycin (AZM) is effective in cystic fibrosis (CF) patients . However, the underlying mechanisms remain uncertain . Some macrolides, including AZM, display inhibition of virulence factors and other antipseudomonal effects at subinhibitory levels in vitro . OBJECTIVES: Drug doses used for CF and DPB therapy were investigated to determine whether they achieve corresponding sputum drug levels in CF patients in vivo . METHODS: In an open, prospective study, 14 CF patients with chronic Pseudomonas aeruginosa airway infection received 250 mg AZM either daily ('high dose') or twice weekly ('low dose') for 12 weeks . Viscoelasticity of sputum was assessed by magnetic microrheology . RESULTS: AZM accumulated in sputum by two orders of magnitude over a period of four weeks . In the following steady state, median AZM concentrations in sputum were 9.5 microg/mL (0.6 to 79.3 microg/mL, interquartiles 1.4 to 33.4 microg/mL) and 0.5 microg/mL (range less than 0.1 {below detection level} to 5.2 microg/mL, interquartiles 0.2 to 1.4 microg/mL) in the high and low dose groups, respectively . Viscoelasticity improved in all patients but one . CONCLUSIONS: The findings suggest that antipseudomonal activity has to be considered among the potential mechanisms of macrolide therapy . Further, viscoelasticity may be a valuable parameter in future clinical trials.

J Mol Microbiol Biotechnol, 2003, 6(2), 88 - 100
The role of regulators in the expression of quorum-sensing signals in Pseudomonas aeruginosa; Fagerlind MG et al.; Quorum-sensing systems provide Pseudomonas aeruginosa with a sensitive regulatory mechanism that allows for the induction of several phenotypic genes in a cell density fashion . In this work, a mathematical model of the acylated homoserine lactones regulatory network system in P . aeruginosa has been developed . It is the first integrated model to consider both quorum-sensing systems . The model has allowed us to disentangle the complex behavior exhibited by the system as the concentration of extracellular OdDHL is increased . At either low or high levels of extracellular OdDHL, the bacterium remains in an uninduced or induced state, respectively . At moderate levels, the behavior is characterized by several states . Here, the bacteria can switch suddenly from an uninduced to an induced phenotype in response to small changes in the concentration of extracellular OdDHL . Additionally, we have been able to address the roles of RsaL and Vfr as regulators of the quorum-sensing system . An important result from this analysis suggests that RsaL will increase the concentration of extracellular OdDHL required to induce the system, and it is a key regulator of the inhibition of the quorum-sensing system under low cell densities . Most importantly, our results suggest that Vfr has strong regulatory effects on the system as an increased affinity between the LasR/OdDHL complex, and the lasR promoter leads to significant qualitative changes in induction patterns . We also show experimental data that demonstrate that Vfr is required for signal production in the early phase of growth, but that in the latter stages of growth, the vfr mutant is able to synthesize wild-type levels of signal .

Di Yi Jun Yi Da Xue Xue Bao, 2004 Mar, 24(3), 303 - 5
{Detection of oprI gene of Pseudomonas aeruginosa by reverse dot-blot hybridization}; Fan HZ et al.; OBJECTIVE: To establish a rapid method for detecting Pseudomonas aeruginosa at the early stage of infection . METHODS: Specific primers were designed according to oprI gene sequence of Pseudomonas aeruginosa, and the specific probe was synthesized by PCR . After photosensitive biotin labeling of the bacterial DNA, reverse dot-blot hybridization was used to detect Pseudomonas aeruginosa . RESULTS: The probe synthesized was highly specific to Pseudomonas aeruginosa without cross reaction with other bacteria, viruses or fungi . The method was capable of detecting 100 ng bacteria DNA . CONCLUSION: Reverse dot-blot hybridization possesses the merits of speediness and specificity in the detection of Pseudomonas aeruginosa in the early stage of infection.

Vaccine, 2004 Feb 17, 22(7), 840 - 7
Recombinant OprF-OprI as a vaccine against Pseudomonas aeruginosa infections; Baumann U et al.; A vaccine against Pseudomonas aeruginosa based on recombinant outer membranes has been developed . After intramuscularly injecting into patients with severe burns, antibodies against P . aeruginosa were induced . Vaccination was well tolerated . Intranasal application of the vaccine into volunteers, induced specific s-IgA antibodies . We conclude that the newly developed vaccine may be suitable for protection of the main risk groups of P . aeruginosa infections . In particular, for the protection of burn patients and patients with cystic fibrosis.

Vaccine, 2004 Feb 17, 22(7), 831 - 9
Pseudomonas immunotherapy: a historical overview; Holder IA; The historic development of vaccines to be used as immunotherapy for Pseudomonas aeruginosa infections, in various patient populations, is reviewed . Commentary is offered concerning the relevance of each approach in light of our current understanding of the pathological process of these infections.

Respir Res . 2004 Feb 12;5(1):1.
TLR4 signaling is essential for survival in acute lung injury induced by virulent Pseudomonas aeruginosa secreting type III secretory toxins; Faure K et al.; BACKGROUND: The relative contributions of the cytotoxic phenotype of P . aeruginosa expressing type III secretory toxins and an immunocompromised condition lacking normal Toll-like receptor 4 (TLR4) signaling in the pathogenesis of acute lung injury and sepsis were evaluated in a mouse model for Pseudomonas aeruginosa pneumonia . By using lipopolysaccharide-resistant C3H/HeJ mice missing normal TLR4 signaling due to a mutation on the tlr4 gene, we evaluated how TLR4 signaling modulates the pneumonia caused by cytotoxic P . aeruginosa expressing type III secretory toxins . METHODS: We infected C3H/HeJ or C3H/FeJ mice with three different doses of either a cytotoxic P . aeruginosa strain (wild type PA103) or its non-cytotoxic isogenic mutant missing the type III secretory toxins (PA103DeltaUT) . Survival of the infected mice was evaluated, and the severity of acute lung injury quantified by measuring alveolar epithelial permeability as an index of acute epithelial injury and the water to dry weight ratios of lung homogenates as an index of lung edema . Bacteriological analysis and cytokine assays were performed in the infected mice . RESULTS: Development of acute lung injury and sepsis was observed in all mouse strains when the cytotoxic P . aeruginosa strain but not the non-cytotoxic strain was instilled in the airspaces of the mice . Only C3H/HeJ mice had severe bacteremia and high mortality when a low dose of the cytotoxic P . aeruginosa strain was instilled in their lungs . CONCLUSION: The cytotoxic phenotype of P . aeruginosa is the critical factor causing acute lung injury and sepsis in infected hosts . When the P . aeruginosa is a cytotoxic strain, the TLR4 signaling system is essential to clear the bacteria to prevent lethal lung injury and bacteremia.

Infect Immun, 2004 Apr, 72(4), 2045 - 51
Role for cystic fibrosis transmembrane conductance regulator protein in a glutathione response to bronchopulmonary pseudomonas infection; Day BJ et al.; The lung maintains an elevated level of glutathione (GSH) in epithelial lining fluid (ELF) compared to serum . The mechanism(s) by which the lung maintains high levels of ELF GSH and factors that modulate them are largely unexplored . We hypothesized that lung cystic fibrosis transmembrane conductance regulator protein (CFTR) modulates GSH efflux in response to extracellular stress, which occurs with lung infections . Mice were challenged intratracheally with Pseudomonas aeruginosa, and on the third day of infection bronchoalveolar lavage fluid was obtained and analyzed for cytokines and antioxidants . Lung tissue antioxidants and enzyme activities were also assessed . P . aeruginosa lung infection increased levels of inflammatory cytokines and neutrophils in the ELF . This corresponded with a marked threefold increase in GSH and a twofold increase in urate levels in the ELF of P . aeruginosa-infected wild-type mice . A twofold increase in urate levels was also observed among lung tissue antioxidants of P . aeruginosa-infected wild-type mice . There were no changes in markers of lung oxidative stress associated with the P . aeruginosa lung infection . In contrast with wild-type mice, the CFTR knockout mice lacked a significant increase in ELF GSH when challenged with P . aeruginosa, and this correlated with a decrease in the ratio of reduced to oxidized GSH in the ELF, a marker of oxidative stress . These data would suggest that the lung adapts to infectious agents with elevated ELF GSH and urate . Individuals with lung diseases associated with altered antioxidant transport, such as cystic fibrosis, might lack the ability to adapt to the infection and present with a more severe inflammatory response.

Invest Ophthalmol Vis Sci, 2004 Apr, 45(4), 1182 - 7
Murine ocular heparanase expression before and during infection with Pseudomonas aeruginosa; Berk RS et al.; PURPOSE: To demonstrate the constitutive expression and regulation of heparanase (heparan sulfate endoglycosidase) in the normal mouse eye and in mice intracorneally infected with Pseudomonas aeruginosa . METHODS: Naive (unimmunized) and immunized C57BL/6J mice were infected with P . aeruginosa, and corneal heparanase gene and protein expression were detected by semiquantitative RT-PCR and immunoblot analysis . Immunohistochemistry was also applied to characterize corneal heparanase in naive mice . RESULTS: Heparanase mRNA and protein expression were detected in uninfected corneas of C57BL/6J mice . Immunohistochemical studies indicated heparanase protein expression was primarily in the corneal epithelium before corneal infection and was also in the corneal stroma after infection . Immunohistochemical studies of uninfected and infected whole eyes of naive mice indicated heparanase protein expression in most layers of the retina, but the expression did not appear to be upregulated during corneal infection . Staining was most intense in the inner photoreceptor layer of the retina . CONCLUSIONS: Heparanase was constitutively expressed in both the corneal epithelium and several retinal layers before intracorneal infection with P . aeruginosa . Temporal upregulation of corneal heparanase protein expression was detected in naive mice during infection, most likely due to heparanase positive infiltrating cells, but the protein was not upregulated in corneas from immunized mice because they had a lower inflammatory response, associated with the restoration of corneal clarity . There did not appear to be temporal upregulation of heparanase expression in the retina of infected mice, as determined by immunohistochemistry.

Curr Opin Microbiol, 2004 Feb, 7(1), 39 - 44
Analysis of regulatory networks in Pseudomonas aeruginosa by genomewide transcriptional profiling; Goodman AL et al.; Transcriptional profiling using DNA microarrays has proved to be a valuable tool for dissecting bacterial adaptation to various environments, including human hosts . Analysis of genomes and transcriptomes of Pseudomonas aeruginosa shows that this bacterium possesses and expresses a core set of genes, including virulence factors, which allow it to thrive in a range of environments . Transcriptional regulators previously thought to control single virulence traits are now shown to regulate complex global signaling networks . Microarray-based research has led to the discovery of upstream regulators and downstream components of these pathways, as well as probed the response to antibiotics, environmental stresses and other bacteria . Independent studies have highlighted the role of media composition, the makeup of the physical environment and experimental methods in the outcome of microarray analyses . A compilation of all the published data clearly shows transcriptional regulation of genes in all functional classes . Under conditions examined to date, slightly more than a quarter of the genome is regulated, suggesting that P . aeruginosa may use much of its genome for conditions unexplored in the laboratory.

J Cataract Refract Surg, 2004 Feb, 30(2), 457 - 63
Folding procedure for acrylic intraocular lenses; Mencucci R et al.; PURPOSE: To compare in vitro the effect of 2 standard methods of folding acrylic intraocular lenses (IOLs) on surface characteristics and bacterial adhesion . SETTING: Eye Clinic and Department of Health-Microbiology Unit, University of Florence, Florence, Italy . METHODS: To evaluate the effect of folding, 2 types of acrylic IOLs were not folded or folded with a forceps or an injector and then processed for scanning electron microscopy (SEM) examination . Bacterial adhesion was assessed using an ocular isolate of Pseudomonas aeruginosa . Nonfolded and folded IOLs were placed in test tubes containing the bacterial suspension for direct counting of viable adherent bacteria and for SEM . RESULTS: The injector-folded IOLs did not show major alterations on the surface; 5 of the 9 forceps-folded IOLs showed marks or scratches in the profile of the optic . The mean number of viable adherent bacteria per area of IOL optic was 1082 (95% confidence interval {CI}, 835-1330) in forceps-folded IOLs, 366 (95% CI, 192-359) in injector-folded IOLs, and 206 (95% CI, 123-289) in nonfolded IOLs . Scanning electron microscopy confirmed more surface irregularities on forceps-folded IOLs, with bacteria adherent preferentially on the surface scratches . CONCLUSION: Forceps-folding provoked more surface irregularities, which probably make IOLs more susceptible to bacterial adhesion.

Med Clin (Barc), 2004 Mar 6, 122(8), 311 - 6
{Azithromycin therapy in cystic fibrosis}; Maiz Carro L et al.; Progressive lung disease, caused by chronic endobronchial colonization, is the major cause of morbidity and mortality in patients with cystic fibrosis (CF) . Several pathogens, including Staphylococcus aureus and Pseudomonas aeruginosa are responsible for this effect . The steadily improving prognosis of CF has been attributed to the use of antibiotics with activity against these organisms . Despite a significant increase in the amount of published material demonstrating the potential role of macrolide antibiotics as antiinflammatory agents and their effects on bacterial virulence, their mechanism of action in CF patients is still unknown . Although there is a limited number of clinical trials assessing the efficacy and safety of azithromycin (AZM) in CF, increasing evidence suggests that 3 to 6-month AZM treatment in CF patients is safe and well tolerated . This treatment results in clinical improvement, decreasing the number of pulmonary exacerbations and increasing pulmonary function . Therefore, chronic treatment with AZM should be considered in CF patients added to conventional therapy . Clinical experience with macrolides other than AZM in CF patients is very limited.

Prikl Biokhim Mikrobiol, 2004 Jan-Feb, 40(1), 78 - 82
{Effect of natural and genetically modified rhizospheric Pseudomonas aureofaciens bacteria on accumulation of arsenic by plants}; Sizova OI et al.; Gene constructions rendering bacteria resistant to arsenic and capable of dissolving phosphates and/or arsenates were created by cloning ars operon and the gene of citrate synthase from a chromosome of the strain Pseudomonas aeruginosa PAO1 . Genetically modified variants of the strain Pseudomonas aureofaciens BS1393 have been constructed, which are resistant to high concentrations of arsenic and dissolve poorly soluble phosphates and/or arsenates . Recombinant strains P . aureofaciens BS1393(pUCP22::arsRBC) and P . aureofaciens BS1393(pUCP22::gltA) exerted positive effects on the survival of sorgo (Sorghum saccharatum L.) and its ability to accumulate arsenic.

J Bacteriol, 2004 Apr, 186(7), 2115 - 22
Sequence polymorphism in the glycosylation island and flagellins of Pseudomonas aeruginosa; Arora SK et al.; A genomic island consisting of 14 open reading frames, orfA to orfN was previously identified in Pseudomonas aeruginosa strain PAK and shown to be essential for glycosylation of flagellin . DNA microarray hybridization analysis of a number of P . aeruginosa strains from diverse origins showed that this island is polymorphic . PCR and sequence analysis confirmed that many P . aeruginosa strains carry an abbreviated version of the island (short island) in which orfD, -E and -H are polymorphic and orfI, -J, -K, -L, and -M are absent . To ascertain whether there was a relationship between the inheritance of the short island and specific flagellin sequence variants, complete or partial nucleotide sequences of flagellin genes from 24 a-type P . aeruginosa strains were determined . Two distinct flagellin subtypes, designated A1 and A2, were apparent . Strains with the complete 14-gene island (long island) were almost exclusively of the A1 type, whereas strains carrying the short island were associated with both A1- and A2-type flagellins . These findings indicate that P . aeruginosa possesses a relatively low number of distinct flagellin types and probably has the capacity to further diversify this antigenic surface protein by glycosylation.

Diagn Microbiol Infect Dis, 2004 Mar, 48(3), 181 - 9
Evaluation of an impedance method for subtyping of Pseudomonas aeruginosa; Wu JJ et al.; Eight isolates (no . 1 to 8) of Pseudomonas aeruginosa isolated from 8 burn patients were typed by pulsed-field gel electrophoresis (PFGE), arbitrarily primed polymerase chain reaction (PCR) (AP-PCR), biotyping, antimicrobial susceptibility testing, and a newly developed technique-impedance method . By both PFGE and AP-PCR, isolates 1 and 2 were designated type A, while isolates 3 to 8 were designated type B . However, isolates 3 to 8 could be further divided into three distinct subtypes (B, C, and D) by the impedance method . Four antibiograms were obtained by testing the 8 isolates against six antimicrobial agents and designation of antibiogram to each of the 8 isolates was in accordance with those obtained by the impedance method . The results of biotyping did not agree with any of the above typing methods . In conclusion, the impedance technique had a high discriminatory ability to differentiate genetically related clones into subtypes . The method is simple, reproducible, and has a high typeability.

Curr Opin Infect Dis, 2004 Apr, 17(2), 109 - 12
Role of nebulized antibiotics for the treatment of respiratory infections; Klepser ME; PURPOSE OF REVIEW: The purpose of this review is to summarize modern data pertaining to the use of aerosolized antimicrobials for the treatment of and prophylaxis against pulmonary infections . RECENT FINDINGS: Few recent publications have examined the safety and efficacy of nebulized antibiotics . Two well conducted trials have been published that describe the utility of tobramycin solution for inhalation among cystic fibrosis patients . A couple of good reviews have also been published that have summarized the use of aerosolized antibiotics in other patient populations . SUMMARY: Data regarding this topic are scarce . At this time, data support the use of aerosolized tobramycin solution for inhalation in cystic fibrosis patients infected or colonized by Pseudomonas aeruginosa . Apart from this situation, widespread aerosolized administration of other agents in cystic fibrosis and non-cystic fibrosis patient populations should not be advocated.

Am J Physiol Lung Cell Mol Physiol, 2004 Jul, 287(1), L94 - 103 Epub 2004 Mar 12.
Pseudomonas aeruginosa pyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells; O'Malley YQ et al.; Production of pyocyanin enhances Pseudomonas aeruginosa virulence . Many of pyocyanin's in vitro and in vivo cytotoxic effects on human cells appear to result from its ability to redox cycle . Pyocyanin directly accepts electrons from NADH or NADPH with subsequent electron transfer to oxygen, generating reactive oxygen species . Reduced glutathione (GSH) is an important cellular antioxidant, and it contributes to the regulation of redox-sensitive signaling systems . Using the human bronchial epithelial (HBE) and the A549 human type II alveolar epithelial cell lines, we tested the hypothesis that pyocyanin can deplete airway epithelial cells of GSH . Incubation of both cell types with pyocyanin led to a concentration-dependent loss of cellular GSH (up to 50%) and an increase in oxidized GSH (GSSG) in the HBE, but not A549 cells, at 24 h . An increase in total GSH, mostly as GSSG, was detected in the culture media, suggesting export of GSH or GSSG from the pyocyanin-exposed cells . Loss of GSH could be due to pyocyanin-induced H(2)O(2) formation . However, overexpression of catalase only partially prevented the pyocyanin-mediated decline in cellular GSH . Cell-free electron paramagnetic resonance studies revealed that pyocyanin directly oxidizes GSH, forming pyocyanin free radical and O(2)(-) . Pyocyanin oxidized other thiol-containing compounds, cysteine and N-acetyl-cysteine, but not methionine . Thus GSH may enhance pyocyanin-induced cytotoxicity by functioning as an alternative source of reducing equivalents for pyocyanin redox cycling . Pyocyanin-mediated alterations in cellular GSH may alter epithelial cell functions by modulating redox sensitive signaling events.

Biochem Biophys Res Commun, 2004 Apr 2, 316(2), 323 - 31
Lysophospholipase A activity of Pseudomonas aeruginosa type III secretory toxin ExoU; Tamura M et al.; Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU that is delivered directly into the eukaryotic cell via the type III secretion system . Recent studies demonstrated that ExoU has lipase activity, and that the cytotoxicity of ExoU is dependent on its patatin-like phospholipase domain . We investigated the phospholipase A (PLA) activity of ExoU . ExoU, but not non-catalytic ExoU-S142A, preincubated with the BEAS-2B cell lysate showed a weak increase of Ca(2+)-independent PLA(2) activity . When activated ExoU was mixed with secretory type PLA(2), more phospholipase activity was observed, suggesting that ExoU has lysophospholipase A (lysoPLA) activity . A significant increase in lysoPLA activity was also observed . Glycerol enhanced this activity and inhibitors of iPLA(2) suppressed ExoU's lysoPLA activity . Our results suggest that ExoU has a potent lysoPLA activity that requires the presence of the catalytically active site Ser(142) with an unknown eukaryotic cell factor(s) for its activation.

FEMS Microbiol Lett, 2004 Mar 12, 232(1), 107 - 12
Essential residues in the chromate transporter ChrA of Pseudomonas aeruginosa; Aguilera S et al.; The chrA gene of Pseudomonas aeruginosa plasmid pUM505 encodes the hydrophobic protein ChrA, which confers resistance to chromate by the energy-dependent efflux of chromate ions . Chromate-sensitive mutants were isolated by in vivo random mutagenesis . Transport experiments with cell suspensions of selected mutants showed that 51CrO4(2-) extrusion was drastically lowered as compared to suspensions of the strain with the wild-type plasmid, confirming that the mutations affected a chromate efflux system . DNA sequence analysis showed that most point mutations affected amino acids clustered in the N-terminal half of ChrA, altering either cytoplasmic regions or transmembrane segments, and replaced residues moderately to highly conserved in ChrA homologs . PhoA and LacZ translational fusions were used to confirm the membrane topology at the N-terminal half of the ChrA protein.

Ophthalmology, 2004 Mar, 111(3), 590 - 5
Orthokeratology lens-related corneal ulcers in children: a case series; Young AL et al.; OBJECTIVE: Orthokeratology is a process by which the corneal curvature is flattened by sequentially fitting rigid gas permeable contact lenses of decreasing central curvature . There has been a resurgence of interest with the recent introduction of reverse geometry lenses . Although promising results have been described in reducing the myopic refractive error, the use of these lenses can be associated with corneal problems, as reported in this case series . DESIGN: Observational case series . PARTICIPANTS: Six children with orthokeratology-related corneal ulcers . METHODS: Consecutive cases of orthokeratology lens (OKL)-related corneal ulcers in children presented to a tertiary referral center (March 1999-June 2001) were reviewed . MAIN OUTCOME MEASURES: Preinfection and postinfection visual acuity, refraction, any organisms identified . RESULTS: Six children between the ages of 9 and 14 years (mean = 12.1) were treated . The male:female ratio was 1:5 . All cases were unilateral, with equal numbers of left and right eyes . All children wore the OKL at night for a duration of 8 to 12 hours, with the onset of infection between 3 and 36 months (mean = 16.6) of OKL wear . All of the patients suffered a resultant best-corrected visual acuity loss . Five of the 6 cases were culture positive for Pseudomonas aeruginosa . CONCLUSIONS: In view of the temporary benefits of orthokeratology, together with a known increased risk of infection associated with overnight lens wear, parents of children considering orthokeratology must be informed and warned of the potential for permanent loss of vision . The ophthalmic community should have a heightened awareness of the associated complications.

J Hosp Infect, 2004 Feb, 56(2), 111 - 8
Clinical surveillance of surgical imipenem-resistant Pseudomonas aeruginosa infection in a Japanese hospital; Sasaki M et al.; In order to elucidate any changes in imipenem-resistant Pseudomonas aeruginosa (IRPA) infections in Japan, we examined 511 P . aeruginosa stains isolated from our surgical ward between 1987 and 2001 . These isolates were subjected to susceptibility testing against various antipseudomonal agents including imipenem, meropenem, ceftazidime, gentamicin and ciprofloxacin . They were serotyped with the slide agglutination test and genotyped using pulsed-field gel electrophoresis (PFGE) . The annual incidences of IRPA infections were particularly high in the early 1990s . Epidemiological investigations revealed that these outbreaks were due to dissemination of hospital-acquired IRPA isolates . Intensive use of imipenem promoted the selection of highly resistant strains . Further study of resistance mechanisms revealed that none of the 110 IRPA strains were metallo-beta-lactamase (MBL) producers . Polymerase chain reaction (PCR) analysis using bla(IMP) specific primers confirmed that no IMP-1 type MBL gene-positive strains were detected from our ward . Susceptibilities of those IRPA strains against other antipseudomonal agents showed relatively low levels, suggesting that imipenem resistance was mainly due to impermeability of the OprD porin . In conclusion, hospital-acquired outbreaks of IRPA were recently reduced by guidelines for, and surveillance of, appropriate use of antimicrobial agents . When the rate of IRPA isolation increases, serotyping should be performed initially and PFGE is required to confirm outbreaks . A computer-assisted genotyping technique is available to perform epidemiological studies of IRPA isolates.

Vet Microbiol, 2004 Mar 26, 99(1), 67 - 74
Identification of an alkaline ceramidase gene from Dermatophilus congolensis; Garcia-Sanchez A et al.; A random amplified polymorphic DNA (RAPD) procedure was used to identify a specific 0.6 kb DNA fragment unique to Dermatophilus congolensis . This 0.6 kb fragment was evaluated as a specific DNA probe and used to design oligonucleotide primers for polymerase chain reaction (PCR) amplification . The nucleotide sequences adjacent to this DNA fragment were determined by inverse PCR allowing the identification of a 4.1 kb sequence . Analysis of this revealed a complete open reading frame (ORF) with a high similarity to an alkaline ceramidase from Pseudomonas aeruginosa . The molecular weight of the enzyme derived from the predicted amino acid sequence is 74,662 Da, its pI is 9.81 . The predicted N-terminal sequence of the enzyme contains a signal sequence indicating that the enzyme is exported by the bacterium . Since ceramides have important protective and cell regulatory roles in the epidermis we suggest that this ceramidase may have a role in the pathogenesis of dermatophilosis . It is the first completely sequenced gene described for D . congolensis.

Vet Microbiol, 2004 Apr 5, 99(2), 121 - 9
Pseudomonas aeruginosa from canine otitis externa exhibit a quorum sensing deficiency; Tron EA et al.; Pseudomonas aeruginosa LasB elastase gene (lasB) transcription depends on cell density-dependent quorum-sensing mechanisms of gene activation . Previously, we collected several non-mucoid P . aeruginosa veterinary isolates and showed that the total matrix protease phenotype was similar for isolates regardless of host and site of isolation . In contrast, isolates from chronic canine ear infections (otitis externa) were significantly more likely to exhibit less elastase activity as measured by elastin Congo red than from any other site {Clin . Diag . Lab . Immun . 8 (2001) 632} . In this study, we found that the elastase deficiency phenotype is stable upon passage in broth culture . Transcript amplification analyses indicated that the elastase deficiency appears to be strain-specific, with each isolate exhibiting a unique expression profile relative to strain PAO1 . Although a number of strain-specific transcriptional differences were observed, the overall pattern that emerges is a quorum sensing deficiency among canine ear P . aeruginosa isolates.

Hum Gene Ther, 2004 Mar, 15(3), 273 - 85
Pseudomonas aeruginosa-induced neutrophilic lung inflammation is attenuated by adenovirus-mediated transfer of the heme oxygenase 1 cDNA in mice; Tsuburai T et al.; Heme oxygenase (HO) is well known as the rate-limiting enzyme in the oxidative degradation of heme to biliverdin, carbon monoxide (CO), and iron . Based on recent evidence that overexpression of HO-1 confers protection against various types of cell and tissue injury by regulating apoptotic cell death or cytokine expression profiles, the present study was performed to examine whether the transfer of exogenous HO-1 cDNA in the lung would provide therapeutic effect in a murine model of lung inflammation induced by Pseudomonas aeruginosa . HO-1 overexpression clearly attenuated neutrophil influx and decreased numbers of apoptotic bronchial epithelial cells . Interestingly, the overexpression of Bcl-2, a known antiapoptotic factor, was observed and thought to be the mechanism that inhibits bronchial epithelial cellular apoptosis . It is thus suggested that HO-1 overexpression is useful for treating P . aeruginosa-associated lung inflammation by attenuating neutrophil influx and apoptotic cell death.

Z Naturforsch {C}, 2004 Jan-Feb, 59(1-2), 70 - 4
Rhamnolipid biosurfactants produced by Renibacterium salmoninarum 27BN during growth on n-hexadecane; Christova N et al.; A new strain Renibacterium salmoninarum 27BN was isolated for its capacity to utilize n-hexadecane as sole substrate . Growth on n-hexadecane was accompanied with the production of glycolipid surface active substances detected by surface pressure lowering and emulsifying activity . Glycolipid detection by thin layer chromatography and infrared spectra analyses showed for the first time that Renibacterium salmoninarum 27BN secretes the two rhamnolipids RLL and RRLL typical for Pseudomonas aeruginosa . Growth of Renibacterium salmoninarum 27BN on n-hexadecane depended on the bioavailability of the substrate and the secreted rhamnolipids appeared to be efficient in increasing hexadecane availability for the cells.

J Biol Chem, 2004 May 21, 279(21), 22635 - 42 Epub 2004 Mar 10.
Crystal structure of WbpP, a genuine UDP-N-acetylglucosamine 4-epimerase from Pseudomonas aeruginosa: substrate specificity in udp-hexose 4-epimerases; Ishiyama N et al.; The O antigen of lipopolysaccharide in Gram-negative bacteria plays a critical role in bacterium-host interactions, and for pathogenic bacteria it is a major virulence factor . In Pseudomonas aeruginosa serotype O6 one of the initial steps in O-antigen biosynthesis is catalyzed by a saccharide epimerase, WbpP . WbpP is a member of the UDP-hexose 4-epimerase family of enzymes and exists as a homo-dimer . This enzyme preferentially catalyzes the conversion between UDP-GlcNAc and UDPGalNAc above UDP-Glc and UDP-Gal, using NAD(+) as a cofactor . The crystal structures of WbpP in complex with cofactor and either UDP-Glc or UDP-GalNAc were determined at 2.5 and 2.1 A, respectively, which represents the first structural studies of a genuine UDP-GlcNAc 4-epimerase . These structures in combination with complementary mutagenesis studies suggest that the basis for the differential substrate specificity of WbpP is a consequence of the presence of a pliable solvent network in the active site . This information allows for a comprehensive analysis of the relationship between sequence and substrate specificity for UDP-hexose 4-epimerases and enables the formulation of consensus sequences that predict substrate specificity of UDP-hexose 4-epimerases yet to be biochemically characterized . Furthermore, the examination indicates that as little as one residue can dictate substrate specificity . Nonetheless, phylogenetic analysis suggests that this substrate specificity is an evolutionary and highly conserved property within UDP-hexose 4-epimerases.

J Neurooncol, 2004 Jan, 66(1-2), 197 - 201
Long term survival in a patient with recurrent malignant glioma treated with intratumoral infusion of an IL4-targeted toxin (NBI-3001); Rainov NG et al.; Intratumoral infusion of a recombinant targeted toxin (NBI-3001) consisting of the receptor binding domain of human interleukin 4 (IL-4) and Pseudomonas aeruginosa exotoxin A is an investigational treatment for malignant brain tumors . This 27-year-old male patient presented with a recurrent malignant glioma WHO grade IV after surgery and adjuvant radiation and chemotherapy . The recurrence was treated with intratumoral infusion of NBI-3001 at a dose of 9 microg/ml in 66 ml of infusate . Treatment resulted in long-term survival for 3 years after toxin infusion with a durable tumor response . There were some permanent neurological side effects resulting from toxin infusion . The patient eventually died after a late local recurrence of the known brain tumor . Such clinical evolution of a malignant glioma after a single round of immunotoxin infusion is rather unusual . The late local recurrence may suggest that repeated courses rather than a single infusion of intratumoral toxin are possibly needed for successful long-term tumor control.

Mol Biol Evol, 2004 May, 21(5), 903 - 13 Epub 2004 Mar 10.
The evolutionary history of quorum-sensing systems in bacteria; Lerat E et al.; Communication among bacterial cells through quorum-sensing (QS) systems is used to regulate ecologically and medically important traits, including virulence to hosts . QS is widespread in bacteria; it has been demonstrated experimentally in diverse phylogenetic groups, and homologs to the implicated genes have been discovered in a large proportion of sequenced bacterial genomes . The widespread distribution of the underlying gene families (LuxI/R and LuxS) raises the questions of how often QS genes have been transferred among bacterial lineages and the extent to which genes in the same QS system exchange partners or coevolve . Phylogenetic analyses of the relevant gene families show that the genes annotated as LuxI/R inducer and receptor elements comprise two families with virtually no homology between them and with one family restricted to the gamma-Proteobacteria and the other more widely distributed . Within bacterial phyla, trees for the LuxS and the two LuxI/R families show broad agreement with the ribosomal RNA tree, suggesting that these systems have been continually present during the evolution of groups such as the Proteobacteria and the Firmicutes . However, lateral transfer can be inferred for some genes (e.g., from Firmicutes to some distantly related lineages for LuxS) . In general, the inducer/receptor elements in the LuxI/R systems have evolved together with little exchange of partners, although loss or replacement of partners has occurred in several lineages of gamma-Proteobacteria, the group for which sampling is most intensive in current databases . For instance, in Pseudomonas aeruginosa, a transferred QS system has been incorporated into the pathway of a native one . Gene phylogenies for the main LuxI/R family in Pseudomonas species imply a complex history of lateral transfer, ancestral duplication, and gene loss within the genus.

Int J Antimicrob Agents, 2004 Feb, 23(2), 155 - 63
Bacteraemia in Europe--antimicrobial susceptibility data from the MYSTIC surveillance programme; Unal S et al.; The in vitro antimicrobial susceptibility of organisms isolated from bacteraemic versus non-bacteraemic patients was evaluated using data (1997-2001) from the Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Programme . Minimum inhibitory concentration values and susceptibility breakpoints of meropenem and other broad-spectrum antimicrobials were determined using standard methodology . Three thousand one hundred and thirty-six blood culture (BC) isolates and 17261 non-BC isolates were obtained from 51 European MYSTIC centres . Gram-positive bacteria appeared to be more prevalent in BC isolates compared with other sources . Escherichia coli, methicillin-susceptible Staphylococcus aureus and Pseudomonas aeruginosa were isolated most frequently . Antimicrobial susceptibility of isolates from bacteraemic versus non-bacteraemic patients was similar . Meropenem and imipenem were the most active agents against the majority of the Gram-positive and Gram-negative organisms . Ceftazidime, gentamicin and ciprofloxacin generally exhibited the lowest activities against the most commonly isolated organisms . Meropenem was most active against P . aeruginosa and showed the highest potency and activity against all extended-spectrum and AmpC beta-lactamase producers . These results are relevant to the choice of initial, empirical therapy for patients with suspected bacteraemia.

Bioorg Med Chem Lett, 2004 Mar 22, 14(6), 1477 - 81
Identification of novel potent bicyclic peptide deformylase inhibitors; Molteni V et al.; Screening of our compound collection using Staphylococcus aureus Ni-Peptide deformylase (PDF) afforded a very potent PDF inhibitor with an IC(50) in the low nanomolar range but with poor antibacterial activity (MIC) . Three-dimensional structural information obtained from Pseudomonas aeruginosa Ni-PDF complexed with the inhibitor suggested the synthesis of a variety of analogues that would maintain high binding affinity while attempting to improve antibacterial activity . Many of the compounds synthesized proved to be excellent PDF-Ni inhibitors and some showed increased antibacterial activity in selected strains.

Biotechnol Lett, 2004 Jan, 26(1), 35 - 9
Rhamnolipid production by Pseudomonas aeruginosa immobilised in polyvinyl alcohol beads; Jeong HS et al.; A marine bacterium, Pseudomonas aeruginosa BYK-2 (KCTC 18012P), was immobilised by entrapment in 10% (w/v) polyvinyl alcohol beads and optimized for the continuous production of rhamnolipid . The relative activity of rhamnolipid production was maintained at 80 approximately 90% of the initial production during 15 cycles in a repeated batch culture . Continuous culture was performed in a 1.8 1 airlift bioreactor, yielding 0.1 g rhamnolipid h(-1) at a dilution rate of 0.0 18 h(-1), 25 degrees C, initial pH 7, and 0.5 vvm aeration rate with a 1.21 working volume.

Eur J Clin Pharmacol, 2004 Apr, 60(2), 67 - 74 Epub 2004 Mar 05.
Early antibiotic treatment of pseudomonas aeruginosa colonisation in cystic fibrosis: a critical review of the literature; Marchetti F et al.; AIM: To assess the evidence supporting early antibiotic treatment in asymptomatic cystic fibrosis (CF) patients colonised by Pseudomonas aeruginosa (PA) . METHODS: We carried out a computerised (Medline, Embase) and hand search of journals for suitable publications . All English-language clinical studies regarding the efficacy of early antibiotic treatment on PA colonisation in asymptomatic patients were considered . Each eligible publications fitting these criteria were assessed for the following outcome measures: frequency of positive PA cultures; serum level of precipitating antibodies; lung function; survival; number of hospitalisations; adverse effects and resistance to antibiotics . RESULTS: Of the 11 studies eventually considered, 3 were randomised-2 versus placebo- and 8 were cohort studies-2 of which had historical controls . Overall, 309 patients (population range 7-91 patients) were recruited . There was a high variability between the individual studies for age, outcome measures, duration of follow-up (1 to 44 months) and treatment (three studies used only aerosol tobramycin, one colistin, four aerosol colistin plus oral ciprofloxacin, one used intravenous treatment and two miscellaneous therapy) . An overall evaluation indicated that early antibiotic treatment can reduce the number of positive cultures and the anti-PA antibody titre . In one study, FEV1 was better in the treated group (oral ciprofloxacin and nebulised colistin) than in historical controls, while in one placebo-controlled trial, no effect on lung function was shown after 1 year of tobramycin inhalation . Collateral effects and bacterial resistance were not increased . The short follow-up did not allow definite conclusions with regard to the long-term progression of respiratory insufficiency or survival . CONCLUSIONS: Evidence was found that antibiotic treatment can reduce the rate of positive cultures and of anti-PA antibody titres in asymptomatic CF patients with newly isolated PA . Different therapeutic options have not been directly compared: a multi-centre comparative study needs to be carried out.

J Immunol, 2004 Mar 15, 172(6), 3377 - 81
Cutting edge: myeloid differentiation factor 88 is essential for pulmonary host defense against Pseudomonas aeruginosa but not Staphylococcus aureus; Skerrett SJ et al.; Myeloid differentiation factor 88 (MyD88) is an adapter molecule required for signal transduction via Toll-like receptors (TLRs) and receptors of the IL-1 family . Consequently, MyD88-deficient mice are highly susceptible to bacterial infections, including systemic infection with Staphylococcus aureus . To determine the role of MyD88 in innate immunity to bacterial pneumonia, we exposed MyD88-deficient and wild-type mice to aerosolized Pseudomonas aeruginosa or S . aureus . As predicted, MyD88-deficient mice failed to mount an early cytokine or inflammatory response or to control bacterial replication after infection with P . aeruginosa, which resulted in necrotizing pneumonia and death . By contrast, MyD88-deficient mice controlled S . aureus infection despite blunted local cytokine and inflammatory responses . Thus, whereas MyD88-dependent signaling is integral to the initiation of cytokine and inflammatory responses to both pathogens following infection of the lower respiratory tract, MyD88 is essential for innate immunity to P . aeruginosa but not S . aureus.

Am J Epidemiol, 2004 Mar 15, 159(6), 537 - 46
Association between initial disease presentation, lung disease outcomes, and survival in patients with cystic fibrosis; Lai HJ et al.; This US study was conducted to determine whether mode of diagnosis and initial disease presentation influence lung disease and survival in patients with cystic fibrosis . The study population included 27,703 patients reported to the 1986-2000 Cystic Fibrosis Foundation Registry . Patients were segregated into four diagnostic categories: meconium ileus (MI), prenatal/neonatal screening (SCREEN), positive family history (FH), and symptoms other than meconium ileus (SYMPTOM) . When compared with patients in the SCREEN group, those in the MI or SYMPTOM group were found to have significantly greater risks of shortened survival, Pseudomonas aeruginosa acquisition, and forced expiratory volume in 1 second (FEV(1)) below 70% of predicted . In the SYMPTOM group, the greatest risks of shortened survival, P . aeruginosa acquisition, and FEV(1) <70% occurred for patients presenting with combined respiratory and gastrointestinal symptoms, followed by respiratory or gastrointestinal symptoms alone; the best outcomes were in patients with other presenting features . Additionally, patients with presumably "severe" genotypes (DeltaF508 plus other class I, II, III mutations in both alleles) had greater risks of shortened survival and P . aeruginosa acquisition compared with patients with presumably "mild" genotypes (class IV or V mutations in one or both alleles).

Peptides, 2004 Jan, 25(1), 11 - 7
A ribonuclease with antimicrobial, antimitogenic and antiproliferative activities from the edible mushroom Pleurotus sajor-caju; Ngai PH et al.; A 12 kDa ribonuclease preferential for poly U and with much lower activity toward poly A, poly G and poly C was isolated from fresh fruiting bodies of the mushroom Pleurotus sajor-caju . A purification procedure involving ion exchange chromatography on CM-cellulose, affinity chromatography on Red-Sepharose and Heparin-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75 was used . The ribonuclease was adsorbed on all of the first three types of chromatographic media . It exhibited some activity toward herring sperm DNA and calf thymus DNA . The ribonuclease activity was unaffected in the presence of KCl (10 and 100 mM) and NaCl (100 mM and 1 M), but was strongly inhibited by CuSO4 (0.01 and 0.1 mM) and less potently inhibited by other divalent salts including MgCl2, CaCl2, ZnCl2, ZnSO4 and FeSO4 . The optimal pH was 5.5 and the ribonuclease was stable up to 60 degrees C for 1 h . The ribonuclease inhibited mycelial growth in the fungi Fusarium oxysporum and Mycosphaerella arachidicola with an IC50 value of 95 and 72 microM, respectively . Out of the 12 species of bacteria tested, only Pseudomonas aeruginosa and Staphylococcus aureus were inhibited in growth by the ribonuclease . Viability of the tumor cells HepG2 (hepatoma) and L1210 (leukemia) was reduced with an IC50 of 0.22 and 0.1 microM, respectively in the presence of the ribonuclease . The ribonuclease inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 158 nM and 3H-methyl-thymidine uptake by murine splenocytes with an IC50 of 65 nM.

Microb Drug Resist, 2003 Winter, 9(4), 323 - 8
Role of efflux pumps and mutations in genes for topoisomerases II and IV in fluoroquinolone-resistant Pseudomonas aeruginosa strains; Oh H et al.; We have investigated the occurrence of mutations in topoisomerase II (DNA gyrase) subunit B(gyrB) and topoisomerase IV subunit E(parE) and the hyperexpression of genes for four efflux pump proteins in 20 previously described, fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa . Amino acid alterations were found in GyrB in five strains and in ParE in three strains with MIC of norfloxacin > or = 8 mg/L, and it is likely that some of the alterations contribute to the quinolone resistance exhibited by these strains . Seventeen of the 20 strains overproduced mRNA for one or more pump proteins (MexB, MexD, MexF, or MexY), which caused multidrug resistance phenotype in more than half of strains . Two strains were hypermutable and one of them was highly resistant, but the other strain was only moderately resistant.

Biochemistry (Mosc), 2004 Feb, 69(2), 170 - 5
Full structure of the lipopolysaccharide of Pseudomonas aeruginosa immunotype 5; Bystrova OV et al.; The lipopolysaccharide (LPS) of the opportunistic human pathogen Pseudomonas aeruginosa immunotype 5 was delipidated by mild acid hydrolysis, and the products were separated by high-performance anion-exchange chromatography and analyzed by ESI MS and NMR spectroscopy . LPS species of three types were found, including those with an unsubstituted core and the core substituted with one O-polysaccharide repeating unit or with an O-polysaccharide of a variable number of repeating units . The core region is highly phosphorylated, the major species containing two monophosphate groups and one ethanolamine diphosphate group . Based on these and published data on the O-polysaccharide structure, the full structure of the LPS of P . aeruginosa immunotype 5 was established.

J Biomed Mater Res A, 2004 Apr 1, 69(1), 79 - 90
Poly(ethylene glycol)-polyacrylate copolymers modified to control adherent monocyte-macrophage physiology: interactions with attaching Staphylococcus epidermidis or Pseudomonas aeruginosa bacteria; Wagner VE et al.; The ability of various surface modifications of poly(ethylene glycol)-graft-polyacrylate (PEG-g-PA) copolymers (tethered adhesion peptides and fragments of monoclonal antibodies) to modulate monocyte-macrophage cell interactions with surface colonizing bacteria is reported . The PEG-g-PA copolymers were made to inhibit nonspecific protein and cellular adhesion . The copolymers were then covalently modified with either cell adhesion peptides (YRGDS, YEILDV, or YRGES) or fragments of antibodies to monocyte-macrophage integrin receptors (anti-VLA4, anti-beta(1), anti-beta(2), and anti-CD64), which are known to enhance macrophage adhesion and perhaps modulate their activation . Cytokine expression and phagocytosis response by surface adherent monocyte-macrophages to Staphylococcus epidermidis and Pseudomonas aeruginosa bacteria were quantified . The cytokine expression (interleukins 6 and 1 beta) of adherent macrophages in response to the modified polymers only and to bacterial challenges were quantified by dynamic ELISA assays . The adherent macrophage phagocytic response (oxidative burst) to various materials is compared to oxidative responses to both opsonized and nonopsonized S . epidermidis and P . aeruginosa bacteria . The efficiency of adherent macrophages to ingest and kill both species was determined using radiolabeled and fluorescent labeled bacterial cell ingestion studies as a function of the PEG-g-PA surface modification . Materials modified with adhesion peptides marginally enhanced (2x) macrophage attachment versus controls but, upon bacterial challenges, these materials predisposed adherent macrophages to overexpress proinflammatory cytokines and to exhibit a significant phagocytic response . Conversely, PEG-g-PA materials modified by fragments of monoclonal antibodies significantly enhanced (7x) macrophage adhesion but, upon bacterial challenge, "per cell" cytokine expression levels were reduced compared to peptide modified materials . Macrophages adhering to antibody fragment modified surfaces also exhibited sustained enhanced phagocytic response and higher bacterial killing efficiencies when compared with peptide modified materials .

Jpn J Thorac Cardiovasc Surg, 2004 Feb, 52(2), 84 - 7
Transposition of modified latissimus dorsi musculocutaneous flap in the treatment of persistent bronchopleural fistula after posterolateral incision; Hanaoka T et al.; The condition of a 51-year-old man was complicated with empyema and bronchopleural fistula (BPF) after left upper lobectomy and thoracoplasty for pulmonary aspergillosis . On the postoperative day (POD) 12, the opened bronchial stump was directly closed and covered with a pedicled pectoralis major muscle flap . On POD 66, an open-window thoracostomy was done, because of empyema with Pseudomonas aeruginosa Two years later, we could fill the empyema cavity, and close the multiple BPFs with the transposition of a modified pedicled musculocutaneous (MC) flap and the additional thoracoplasty to gain good quality of life . Although the MC flap was a proximal part of the latissimus dorsi muscle, which was dissected along the posterolateral incision of the first operation, it could be successfully transplanted to cover the BPFs in the open-window . In some patients with a small open-window on the upper anterior chest wall, the pedicled proximal latissimus dorsi MC flap may be very useful for treating persistent BPFs even after a standard posterolateral incision.

BMJ . 2004 Mar 20;328(7441):668 . Epub 2004 Mar 02.
Beta lactam monotherapy versus beta lactam-aminoglycoside combination therapy for sepsis in immunocompetent patients: systematic review and meta-analysis of randomised trials; Paul M et al.; OBJECTIVE: To compare beta lactam monotherapy with beta lactam-aminoglycoside combination therapy for severe infections . DATA SOURCES: Medline, Embase, Lilacs, Cochrane Library, and conference proceedings, to 2003; references of included studies; contact with all authors . No restrictions, such as language, year of publication, or publication status . STUDY SELECTION: All randomised trials of beta lactam monotherapy compared with beta lactam-aminoglycoside combination therapy for patients without neutropenia who fulfilled criteria for sepsis . DATA SELECTION: Two reviewers independently applied selection criteria, performed quality assessment, and extracted the data . The primary outcome assessed was all cause fatality by intention to treat . Relative risks were pooled with the random effect model (relative risk < 1 favours monotherapy) . RESULTS: 64 trials with 7586 patients were included . There was no difference in all cause fatality (relative risk 0.90, 95% confidence interval 0.77 to 1.06) . 12 studies compared the same beta lactam (1.02, 0.76 to 1.38), and 31 studies compared different beta lactams (0.85, 0.69 to 1.05) . Clinical failure was more common with combination treatment overall (0.87, 0.78 to 0.97) and among studies comparing different beta lactams (0.76, 0.68 to 0.86) . There was no advantage to combination therapy among patients with Gram negative infections (1835 patients) or Pseudomonas aeruginosa infections (426 patients) . There was no difference in the rate of development of resistance . Nephrotoxicity was significantly more common with combination therapy (0.36, 0.28 to 0.47) . Heterogeneity was not significant for these comparisons . CONCLUSIONS: In the treatment of sepsis the addition of an aminoglycoside to beta lactams should be discouraged . Fatality remains unchanged, while the risk for adverse events is increased.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 518 - 20 Epub 2004 Feb 25.
Crystallization of Pseudomonas aeruginosa AHL synthase LasI using beta-turn crystal engineering; Gould TA et al.; In Gram-negative bacteria, intercellular communication and virulence regulation is mediated by the diffusible chemical signal acyl-homoserine-L-lactone (AHL) . The AHL synthase enzymes produce a variety of AHLs from the substrates S-adenosyl-L-methionine and acyl-acyl carrier protein . LasI, the AHL synthase from Pseudomonas aeruginosa, has low solubility and has failed to crystallize despite extensive crystallization trials . Based on the previously determined structure of the AHL synthase EsaI, active soluble LasI was produced by re-engineering residues in a tight turn to produce a type I' beta-turn . The resulting protein is active, more stable than the wild-type LasI and has been crystallized in the cubic space group F23, with unit-cell parameters a = b = c = 154.90 A.

Microbiology, 2004 Mar, 150(Pt 3), 539 - 47
The truA gene of Pseudomonas aeruginosa is required for the expression of type III secretory genes; Ahn KS et al.; Invasive strains of Pseudomonas aeruginosa can cause rapid host cell apoptosis by injecting the type III effector molecule ExoS . A transposon insertional mutant bank of P . aeruginosa was screened to identify P . aeruginosa genes that contribute to the ability of the bacteria to trigger host cell apoptosis . Several isolated mutants had disruptions in the fimV gene . A fimV mutant was unable to induce the expression of exoS, exoT and exsA genes under type III inducing conditions, thus exhibiting a defect in type III protein secretion . Furthermore, this mutant was defective in twitching motility, although type IV pili were present on the bacterial surface . Complementation by a fimV-containing cosmid clone restored both phenotypes to the wild-type levels . However, expression of the type III genes in the fimV mutant was not restored by the introduction of a fimV gene alone, although it restored the twitching motility . A gene downstream of fimV, encoding a tRNA pseudouridine synthase (truA) homologue, was able to complement the type III gene expression defect of the fimV mutant . Thus fimV and truA form an operon and fimV mutation has a polar effect on truA . Indeed, a truA mutant is defective in type III gene expression while its twitching motility is unaffected, and a truA clone is able to complement the type III secretion defect . Pseudouridination of tRNAs is important for tRNA structure, thereby improving the fidelity of protein synthesis and helping to maintain the proper reading frame; thus the results imply that truA controls tRNAs that are critical for the translation of type III genes or their regulators.

Yao Xue Xue Bao, 2003 Nov, 38(11), 801 - 4
{Inhibitory effect of egg white lysozyme on ceftazidime-induced release of endotoxin from Pseudomonas aeruginosa}; Liang AH et al.; AIM: To investigate the inhibitory effect of egg white lysozyme (LZM) on ceftazidime (CFT)-induced release of endotoxin from Pseudomonas aeruginosa . METHODS: P . aeruginosa PAO1 was inoculated in nutrition broth or diluted rabbit blood free of antibiotics in the presence or absence of LZM and incubated at 37 degrees C on a water bath shaker . beta-Lactam antibiotic, CFT, was added to cultures at 3.5 h (nutrition broth culture) or 5 h (diluted rabbit blood culture) after inoculation . After 3 h of CFT treatment, the supernatants from different bacterial cultures were prepared by centrifuge and the concentrations of endotoxin in the supernatants were measured . The bacterial supernatants were also added to a murine macrophage cell line RAW 264.7 or intravenously injected into carrageenin-sensitized mice . Tumor necrosis factor-alpha (TNF alpha) and nitric oxide (NO) concentrations in RAW 264.7 supernatants or in mouse sera were tested . RESULTS: CFT treatment alone obviously inhibited the growth of P . aeruginosa PAO1 accompanied by strong and rapid bacteriolysis and released relatively high concentration of endotoxin from bacteria both in nutrition broth and in diluted rabbit blood cultures . The bacterial supernatant from CFT treatment alone yielded high concentrations of TNF alpha both in RAW 264.7 cells and in mice and high level of NO in RAW 264.7 cells . Treatment with the combination of LZM and CFT evidently blocked the lysis of bacteria and reduced the release of endotoxin without decreasing bactericidal activity of CFT . TNF alpha and NO productivity of the supernatants prepared from the LZM/CFT combinative treated bacterial cultures were significantly decreased both in RAW 264.7 cells and in mice indicating that the inflammatory activity was reduced . CONCLUSION: LZM can effectively prevent CFT-induced bacteriolysis, endotoxin release and subsequent proinflammatory factor production but without decreasing bactericidal activity of CFT, resulting in the disassociation of bactericidal activity and bacteriolysis . Thus, LZM might be important for preventing endotoxemia in Gram-negative sepsis with the treatment of antibiotics.

Intensive Care Med, 2004 Jun, 30(6), 1204 - 11 Epub 2004 Feb 26.
Apoptosis inhibition in P . aeruginosa-induced lung injury influences lung fluid balance; Le Berre R et al.; OBJECTIVE: Pseudomonas aeruginosa-induced lung injury is characterized not only by the alteration in lung fluid movement but also by apoptosis of lung epithelial and endothelial cells . We studied whether inhibition of apoptosis using a broad spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD.fmk), would affect lung fluid balance in rat P . aeruginosa pneumonia . METHODS: Z-VAD.fmk (3 mg/kg) was administered intravenously simultaneously with P . aeruginosa intratracheal instillation (0.5 ml/kg, 2 x 10(9) CFU/ml) . Apoptosis was evaluated with the TUNEL technique, cytoplasmic oligonucleosome assay, and caspase 3 activation . To evaluate lung permeability, extravascular plasma equivalent (EPE) and lung wet to dry weight ratio (W/D) were measured 4 h after intratracheal instillation of P . aeruginosa . RESULTS: We found an increase of lung apoptosis 4 h after P . aeruginosa instillation: cytoplasmic oligonucleosome assay increased from 3.17+/-0.78 to 26.82+/-4.67 ODx1000/mg of proteins/ml, Z-VAD.fmk administration decreased this parameter to 10.3+/-2.98 ODx1000/mg of proteins/ml . Caspase 3 levels followed the same pattern . Apoptosis involved both epithelial cells and endothelial cells . Endothelial permeability was increased after Pseudomonas instillation: W/D increased from 3.75+/-0.28 in the Co group to 4.42+/-0.23 in the Pn group; EPE was also higher in the Pn group compared with the Co group (0.125+/-0.04 and 0.002+/-0.01 ml, respectively) . Both of these parameters were improved after Z-VAD.fmk administration; W/D decreased to 3.36+/-0.25 and EPE to 0.02+/-0.02 ml . CONCLUSION: Apoptosis occurs in the early phase of P . aeruginosa pneumonia . Administration of Z-VAD.fmk significantly decreases DNA fragmentation and caspase 3 levels . This is associated with an improvement of endothelial permeability and lung fluid balance.

Zhonghua Jie He He Hu Xi Za Zhi, 2004 Jan, 27(1), 31 - 5
{Risk factors and clinical outcomes of nosocomial infections caused by multidrug resistant Pseudomonas aeruginosa}; Cao B et al.; OBJECTIVE: To investigate the risk factors for multi-drug resistant Pseudomonas aeruginosa (MDRP) infections, and the factors related with poor prognosis of P . aeruginosa infections . METHODS: The data of 44 cases of MDRP nosocomial infections were analyzed from Jan, 1999 . to Dec, 2002 in Peking Union Medical Hospital; 68 cases of antibiotic-sensitive P . aeuroginosa infection were randomized as control . T test, chi-square test and Logistic regression analysis were used for statistics . RESULTS: According to univariate analysis, the factors associated with the development of MDRP nosocomial infection were age, APACHE II, co-infection with other pathogens, hospital acquired pneumonia (HAP), mechanical ventilation, COPD, fluoroquinolone and imipenem/meropenem use 15 days before isolation of MDRP . Multivariate logistic regression analysis identified two independent factors: mechanical ventilation and previous imipenem/meropenem use . Of 44 cases of MDRP infections, 24 died, and 20 survived . Univariate analysis revealed that three factors (APACHE II, mechanical ventilation, resistance switch) were associated with clinical prognosis . But a ccording to multivariate logistic regression analysis, only resistance switch was a predictive factor . CONCLUSIONS: Mechanical ventilation and previous imipenem/meropenem use were independent risk factors for MDRP infection . Resistance switch was a predictive factor for the prognosis of MDRP infection.

FEMS Microbiol Lett, 2004 Feb 16, 231(2), 247 - 52
Chemotaxis proteins and transducers for aerotaxis in Pseudomonas aeruginosa; Hong CS et al.; It was previously shown that the chemotaxis gene cluster 1 (cheYZABW) was required for chemotaxis . In this study, the involvement of the same cluster in aerotaxis is described and two transducer genes for aerotaxis are identified . Aerotaxis assays of a number of deletion-insertion mutants of Pseudomonas aeruginosa PAO1 revealed that the chemotaxis gene cluster 1 and cheR are required for aerotaxis . Mutant strains which contained deletions in the methyl-accepting chemotaxis protein-like genes tlpC and tlpG showed decreased aerotaxis . A double mutant deficient in tlpC and tlpG was negative for aerotaxis . TlpC has 45% amino acid identity with the Escherichia coli aerotactic transducer Aer . The TlpG protein has a predicted C-terminal segment with 89% identity to the highly conserved domain of the E . coli serine chemoreceptor Tsr . A hydropathy plot of TlpG indicated that hydrophobic membrane-spanning regions are missing in TlpG . A PAS motif was found in the N-terminal domains of TlpC and TlpG . On this basis, the tlpC and tlpG genes were renamed aer and aer-2, respectively . No significant homology other than the PAS motif was detected in the N-terminal domains between Aer and Aer-2.

Clin Infect Dis, 2004 Mar 1, 38(5), 670 - 7 Epub 2004 Feb 17.
Acquisition of multidrug-resistant Pseudomonas aeruginosa in patients in intensive care units: role of antibiotics with antipseudomonal activity; Paramythiotou E et al.; A matched case-control study was performed to identify risk factors for acquiring multidrug-resistant Pseudomonas aeruginosa (MDRPA) in intensive care unit (ICU) patients during a 2-year period . MDRPA was defined as P . aeruginosa with combined decreased susceptibility to piperacillin, ceftazidime, imipenem, and ciprofloxacin . Thirty-seven patients who were colonized or infected with MDRPA were identified, 34 of whom were matched with 34 control patients who had cultures that showed no growth of P . aeruginosa . Matching criteria were severity of illness and length of ICU stay, with each control patient staying in the ICU for at least as long as the time period between the corresponding case patient's admission to the ICU and the acquisition of MDRPA . Baseline demographic and clinical characteristics and the use of invasive procedures were similar for case patients and control patients . Multivariate analysis identified duration of ciprofloxacin treatment as an independent risk factor for MDRPA acquisition, whereas the duration of treatment with imipenem was of borderline significance . These data support a major role for the use of antibiotics with high antipseudomonal activity, particularly ciprofloxacin, in the emergence of MDRPA.

Gene Ther, 2004 Jun, 11(12), 1011 - 8
Comparison of HPV DNA vaccines employing intracellular targeting strategies; Kim JW et al.; Intradermal vaccination via gene gun efficiently delivers DNA vaccines into dendritic cells (DCs) of the skin, resulting in the activation and priming of antigen-specific T cells in vivo . In the context of DNA vaccines, we previously used the gene gun approach to test several intracellular targeting strategies that are able to route a model antigen, such as the human papillomavirus type-16 (HPV-16) E7, to desired subcellular compartments in order to enhance antigen processing and presentation to T cells . These strategies include the use of the sorting signal of lysosome-associated membrane protein (LAMP-1), Mycobacterium tuberculosis heat-shock protein 70 (HSP70), calreticulin (CRT) and the translocation domain (dII) of Pseudomonas aeruginosa exotoxin A (ETA) . Vaccination with DNA vaccines encoding E7 antigen linked to any of these molecules all led to a significant enhancement of E7-specific CD8(+) T-cell immune responses and strong antitumor effects against an E7-expressing tumor, TC-1 . However, we were interested in identifying the most potent DNA vaccine for our future clinical trials . Thus, we performed a series of experiments to directly compare the potency of the various DNA vaccines . Among the DNA vaccines we tested, we found that vaccination with pcDNA3-CRT/E7 generated the highest number of E7-specific CD8(+) T cells and potent long-term protection and treatment effects against E7-expressing tumors in mice . Interestingly, we observed that pcDNA3-CRT/E7 is also capable of protecting against an E7-expressing tumor with downregulated MHC class I expression, a common feature associated with most HPV-associated cervical cancers . Our data suggest that the DNA vaccine linking CRT to E7 (CRT/E7) may be a suitable candidate for human trials for the control of HPV infections and HPV-associated lesions.

Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2530 - 5
The broad host range pathogen Pseudomonas aeruginosa strain PA14 carries two pathogenicity islands harboring plant and animal virulence genes; He J et al.; The ubiquitous bacterium Pseudomonas aeruginosa is the quintessential opportunistic pathogen . Certain isolates infect a broad range of host organisms, from plants to humans . The pathogenic promiscuity of particular variants may reflect an increased virulence gene repertoire beyond the core P . aeruginosa genome . We have identified and characterized two P . aeruginosa pathogenicity islands (PAPI-1 and PAPI-2) in the genome of PA14, a highly virulent clinical isolate . The 108-kb PAPI-1 and 11-kb PAPI-2, which are absent from the less virulent reference strain PAO1, exhibit highly modular structures, revealing their complex derivations from a wide array of bacterial species and mobile elements . Most of the genes within these islands that are homologous to known genes occur in other human and plant bacterial pathogens . For example, PAPI-1 carries a complete gene cluster predicted to encode a type IV group B pilus, a well known adhesin absent from strain PAO1 . However, >80% of the PAPI-1 DNA sequence is unique, and 75 of its 115 predicted ORF products are unrelated to any known proteins or functional domains . Significantly, many PAPI-1 ORFs also occur in several P . aeruginosa cystic fibrosis isolates . Twenty-three PAPI ORFs were mutated, and 19 were found to be necessary for full plant or animal virulence, with 11 required for both . The large set of "extra" virulence functions encoded by both PAPIs may contribute to the increased promiscuity of highly virulent P . aeruginosa strains, by directing additional pathogenic functions.

JAMA, 2004 Feb 25, 291(8), 981 - 5
Outbreak of Pseudomonas aeruginosa infections caused by commercial piercing of upper ear cartilage; Keene WE et al.; CONTEXT: Sporadic infections following ear piercing are well documented, but common-source outbreaks are rarely recognized . OBJECTIVE: To investigate reports of auricular chondritis subsequent to commercial ear piercing . DESIGN, SETTING, AND SUBJECTS: Outbreak investigation by Oregon public health agencies, including cohort study of persons pierced at a jewelry kiosk in August-September 2000, environmental sampling, and molecular subtyping of isolates . Confirmed cases had Pseudomonas aeruginosa cultured from ear wounds . Suspected cases had signs and symptoms of external ear infection, including drainage of pus or blood for at least 14 days . MAIN OUTCOME MEASURES: Risk factors for infection and comparison of bacterial isolates by molecular subtyping . RESULTS: From 186 piercings in 118 individuals, we identified 7 confirmed P aeruginosa infections and 18 suspected infections . Confirmed cases were 10 to 19 years old . Most were initially treated with antibiotics ineffective against Pseudomonas . Four were hospitalized, 4 underwent incision and drainage surgeries (1 as an outpatient), and several were cosmetically deformed . Upper ear cartilage piercing was more likely to result in either confirmed or suspected infection than was lobe piercing (confirmed: RR undefined, P<.001; suspected: RR, 3.6; 95% confidence interval, 1.5-8.5) . All persons with confirmed infections had their ear cartilage pierced with an open, spring-loaded piercing gun . Patient isolates were indistinguishable by molecular subtyping, and matching isolates were recovered from a disinfectant bottle and nearby sink . At least 1 worker admitted sometimes spraying the disinfectant on the ear studs before piercing . CONCLUSIONS: Ear cartilage piercing is inherently more risky than lobe piercing . Clinicians should respond aggressively to potential auricular chondritis and consider Pseudomonas a possible cause pending culture results.

Antimicrob Agents Chemother, 2004 Mar, 48(3), 843 - 51
Selection of an antibiotic-hypersusceptible mutant of Pseudomonas aeruginosa: identification of the GlmR transcriptional regulator; Ramos-Aires J et al.; Tn501 random mutagenesis was applied to the Pseudomonas aeruginosa wild-type strain PAO1 to select for mutants hypersusceptible to aminoglycoside antimicrobial agents . One such mutant, called 19A, was found to be hypersusceptible to a wide range of antibiotics including aminoglycosides, beta-lactams, fluoroquinolones, colistin, erythromycin, rifampin, and glycopeptides . Light microscopy of the mutant strain revealed abnormal morphology characterized by large, filamentous cells . The drug supersusceptibility of 19A was accompanied by loss of motility, reduced resistance to osmotic and heat shock stress, and impaired growth at low temperatures . The insertion site of the Tn501 transposon in mutant 19A has occurred in an open reading frame (PA5550 according to the PAO1 genome project), whose gene product shows amino acid sequence similarity to the DeoR family of transcriptional repressors . The gene, which we called glmR, is located between the glmS (PA5549) and glmU (PA5552) homologues of E . coli, responsible for the synthesis of UDP-N-acetylglucosamine-1-P, a precursor of both lipopolysaccharide (LPS) and peptidoglycan . We showed that GlmR represses the transcription of the adjacent glmS homologue (PA5549) in P . aeruginosa, possibly affecting the pool of precursors for peptidoglycan and LPS synthesis . To our knowledge GlmR is the first regulator in P . aeruginosa that affects susceptibility to a large variety of antibiotics and is therefore a potential target for novel anti-infective agents.

Oncogene, 2004 Mar 25, 23(13), 2367 - 78
Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer; Punj V et al.; Azurin, a copper-containing redox protein released by the pathogenic bacterium Pseudomonas aeruginosa, is highly cytotoxic to the human breast cancer cell line MCF-7, but is less cytotoxic toward p53-negative (MDA-MB-157) or nonfunctional p53 cell lines like MDD2 and MDA-MB-231 . The purpose of this study was to investigate the underlying mechanism of the action of bacterial cupredoxin azurin in the regression of breast cancer and its potential chemotherapeutic efficacy . Azurin enters into the cytosol of MCF-7 cells and travels to the nucleus, enhancing the intracellular levels of p53 and Bax, thereby triggering the release of mitochondrial cytochrome c into the cytosol . This process activates the caspase cascade (including caspase-9 and caspase-7), thereby initiating the apoptotic process . Our results indicate that azurin-induced cell death stimuli are amplified in the presence of p53 . In vivo injection of azurin in immunodeficient mice harboring xenografted human breast cancer cells in the mammary fat pad leads to statistically significant regression (85%, P = 0.0179, Kruskal-Wallis Test) of the tumor . In conclusion, azurin blocks breast cancer cell proliferation and induces apoptosis through the mitochondrial pathway both in vitro and in vivo, thereby suggesting a potential chemotherapeutic application of this bacterial cupredoxin for the treatment of breast cancer.

Indian J Pediatr, 2004 Jan, 71(1), 21 - 3
Can throat swab after physiotherapy replace sputum for identification of microbial pathogens in children with cystic fibrosis?
Kabra SK, Alok A, Kapil A, Aggarwal G, Kabra M, Lodha R, Pandey RM, Sridevi K, Mathews J.
OBJECTIVE: To compare cultures throat swab after physiotherapy with results of sputum culture in identification of lower airway pathogens in children with cystic fibrosis . METHODS: 387 samples of sputum cough swabs, throat swab and throat swab after physiotherapy were collected from 48 patients of cystic fibrosis and cultured for aerobic bacteria . The results of cultures of cough swabs, throat swab and throat swab after physiotherapy were compared with results of sputum culture . RESULTS: There was good concordance between culture results of sputum and other methods . Over all concordance was 70%, 81% and 92% with cough swab, throat swab and throat swab after physiotherapy . Sensitivity for isolation of Pseudomonas aeruginosa by throat swab, cough swab and throat swab after physiotherapy was 40%, 42% and 82% respectively . Specificity for isolation of Pseudomonas by throat swab, cough swab and throat swab after physiotherapy was 99%, 100% and 99% respectively . Sensitivity for isolation of Staphylococcus aureus by throat swab, cough swab and throat swab after physiotherapy was 57%, 50% and 100% respectively . Specificity for isolation of Staphylococcus by throat swab, cough swab and throat swab after physiotherapy was 99% for all these methods . CONCLUSION: It is concluded that throat swab after physiotherapy in a child with CF can be used reliably for identification of lower airway pathogens.

J Immunol, 2004 Mar 1, 172(5), 3034 - 41
NK cells, but not NKT cells, are involved in Pseudomonas aeruginosa exotoxin A-induced hepatotoxicity in mice; Muhlen KA et al.; Pseudomonas aeruginosa exotoxin A (PEA) causes T cell- and Kupffer cell (KC)-dependent liver injury in mice . TNF-alpha as well as IL-18 and perforin are important mediators of liver damage following PEA injection . In this study, we focus on the role of NK and NKT cells in PEA-induced liver toxicity . Depletion of both NK and NKT cells by injection of anti-NK1.1 Ab as well as depletion of NK cells alone by anti-asialo GM1 Ab protected mice from PEA-induced hepatotoxicity, whereas mice lacking only NKT cells were susceptible . Additionally, we observed infiltration of NK cells, T cells, and neutrophils into liver parenchyma after injection of PEA . The number of NKT cells, however, remained unchanged . The increase in intrahepatic NK cells depended on KCs and the TNF-alpha-dependent up-regulation of the adhesion molecule VCAM-1 in the liver, but not on NKT cells . PEA also augmented the cytotoxicity of hepatic NK cells against typical NK target cells (YAC-1 cells) . This effect depended on KCs, but not on TNF-alpha or NKT cells . Furthermore, only weak expression of MHC class I was detected on hepatocytes, which was further down-regulated in PEA-treated mice . This could explain the susceptibility of hepatocytes to NK cell cytolytic activity in this model . Our results demonstrate that NK cells, activated and recruited independently of NKT cells, contribute to PEA-induced T cell-dependent liver injury in mice.

Infect Immun, 2004 Mar, 72(3), 1677 - 84
An adenylate cyclase-controlled signaling network regulates Pseudomonas aeruginosa virulence in a mouse model of acute pneumonia; Smith RS et al.; Infections caused by the opportunistic pathogen Pseudomonas aeruginosa involve the interplay of several bacterial virulence factors . It has recently been established that the delivery of toxic effector proteins by the type III secretion system is an important virulence mechanism in several animal models . Furthermore, the expression of the type III secretion system and its effectors has been correlated with a poor clinical outcome during human infections . A novel cyclic AMP (cAMP) regulatory network that controls the expression of virulence factors, including the type III secretion system, was examined to determine its contribution to P . aeruginosa colonization and dissemination in a mouse pneumonia model . Mutants lacking the two genome-encoded adenylate cyclases, CyaA and CyaB, and the cAMP-dependent regulator Vfr were examined . Based on the enumeration of bacteria in lungs, livers, and spleens, as well as the assessment of mouse lung pathology, mutations in the cyaB and vfr genes resulted in a more significantly attenuated phenotype than mutations in cyaA . Moreover, in this model, expression of the type III secretion system was essential for lung colonization and pathology . Strains with mutations in the exsA gene, which encodes a type III regulatory protein, or pscC, which encodes an essential component of the secretion apparatus, were also significantly attenuated . Finally, we demonstrate that virulence can be restored in an adenylate cyclase mutant by the overexpression of exsA, which specifically restores expression of the type III secretion system in the absence of a functional cAMP-dependent regulatory network.

Infect Immun, 2004 Mar, 72(3), 1479 - 86
Nutritional effects on host response to lung infections with mucoid Pseudomonas aeruginosa in mice; van Heeckeren AM et al.; In cystic fibrosis, a recessive genetic disease caused by defects in the cystic fibrosis conductance regulator (CFTR), the main cause of death is lung infection and inflammation . Nutritional deficits have been proposed to contribute to the excessive host inflammatory response in both humans and Cftr-knockout mice . Cftr-knockout mice and gut-corrected Cftr-knockout mice expressing human CFTR primarily in the gut were challenged with Pseudomonas aeruginosa-laden agarose beads; they responded similarly with respect to bronchoalveolar lavage cell counts and levels of the acute-phase cytokines tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-6 . Wild-type mice fed the liquid diet used to prevent intestinal obstruction in Cftr-knockout mice had inflammatory responses to P . aeruginosa-laden agarose beads similar to those of wild-type mice fed an enriched solid diet, so dietary effects are unlikely to account for differences between wild-type mice and mice with cystic fibrosis . Finally, since cystic fibrosis patients and Cftr-knockout mice have an imbalance in fatty acids (significantly lower-than-normal levels of docosahexaenoic acid), the effects of specific supplementation with docosahexaenoic acid of wild-type and Cftr-knockout mice on their inflammatory responses to P . aeruginosa-laden agarose beads were tested . There were no significant differences (P = 0.35) in cumulative survival rates between Cftr-knockout mice and wild-type mice provided with either the liquid diet Peptamen or Peptamen containing docosahexaenoic acid . In conclusion, diet and docosahexaenoic acid imbalances alone are unlikely to explain the differences in the host response to lung infections with mucoid P . aeruginosa between mice with cystic fibrosis and their wild-type counterparts.

Acta Otolaryngol, 2004 Jan, 124(1), 13 - 8
Additive effects of toxin exposure and destruction of semicircular canal on cochlear function: an auditory brainstem response study in the rat; Stenqvist M et al.; OBJECTIVE: To ascertain whether the severity of toxin-related hearing loss and the interval between instillation of toxin and surgical trauma affect hearing recovery capacity following semicircular canal (SCC) surgery in the rat . MATERIAL AND METHODS: Twelve rats were injected with Pseudomonas aeruginosa exotoxin A (PaExoA) . Auditory brainstem responses (ABRs) were measured 72 h and 3 weeks later . Depending on the severity of hearing loss, the rats were divided into two groups: those with moderate (Group A; n = 6) and severe (Group B; n = 6) hearing loss . Three rats from Group A were then operated on 3 weeks after toxin exposure and the other three 3 months after instillation of toxin . All Group B rats were operated on after 3 months . RESULTS: In Group A, post-surgical hearing loss recovered to a varying degree but rats in Group B showed little or no hearing recovery capacity . This difference was statistically significant . When the six rats with moderately toxin-affected ears were compared, statistical differences in recovery capacity between those operated on at 3 weeks and at 3 months were also detected . The group with a shorter interval showed significantly less hearing recovery of inner ear function following surgical trauma . CONCLUSION: When the toxin causes severe hearing damage there is no capacity for cochlear recovery following additional surgical trauma . When the rat inner ear is moderately affected by PaExoA, the interval between toxin exposure and SCC destruction plays a significant role in the ultimate hearing outcome . Cochlear recovery potential seems to be weakened in close temporal proximity to toxin exposure, but recovers with the passage of time.

Rev Argent Microbiol, 2003 Oct-Dec, 35(4), 198 - 204
{Optimization of the conditions for electroporation and the addition nisin for Pseudomonas aeruginosa inhibition}; Santi L et al.; A mathematical approach was applied in order to optimize the effect of electroporation by application of pulsed electric fields (PEF) and nisin addition on the inhibition of a strain of Pseudomonas aeruginosa isolated from river sediments . This strain showed to be highly resistant to nisin as only two log cycles reduction of viable cells were obtained in the presence of 84,000 IU/ml nisin . But when a combination of bacteriocin and selected PEF treatment conditions were applied, 4.4-decimal log cycle reduction could be achieved . PEF and nisin interaction seems to be complex, as at lower electric field intensities (i.e., 5 kV/cm) an increment in the number of pulses applied clearly induced a lower inhibitory effect of nisin . At higher PEF intensities (i.e., 11 kV/cm), the inhibitory effect of nisin increased with the number of pulses applied . Results overall, the obtained indicate the possibility of combining PEF and nisin treatments in order to improve the inhibition of resistant microorganisms . The Doehlert experimental design and surface response methodology was an interesting tool to obtain or predict the optimal combination of the stress factors applied.

J Bacteriol, 2004 Mar, 186(5), 1280 - 6
Evidence for a symbiosis island involved in horizontal acquisition of pederin biosynthetic capabilities by the bacterial symbiont of Paederus fuscipes beetles; Piel J et al.; Pederin belongs to a group of antitumor compounds found in terrestrial beetles and marine sponges . It is used by apparently all members of the rove beetle genera Paederus and Paederidus as a chemical defense against predators . However, a recent analysis of the putative pederin biosynthesis (ped) gene cluster strongly suggests that pederin is produced by bacterial symbionts . We have sequenced an extended region of the symbiont genome to gain further insight into the biology of this as-yet-unculturable bacterium and the evolution of pederin symbiosis . Our data indicate that the symbiont is a very close relative of Pseudomonas aeruginosa that has acquired several foreign genetic elements by horizontal gene transfer . Besides one functional tellurite resistance operon, the region contains a genomic island spanning 71.6 kb that harbors the putative pederin biosynthetic genes . Several decayed insertion sequence elements and the mosaic-like appearance of the island suggest that the acquisition of the ped symbiosis genes was followed by further insertions and rearrangements . A horizontal transfer of genes for the biosynthesis of protective substances could explain the widespread occurrence of pederin-type compounds in unrelated animals from diverse habitats.

J Bacteriol, 2004 Mar, 186(5), 1258 - 69
Differential impact of MexB mutations on substrate selectivity of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa; Middlemiss JK et al.; The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems . To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a DeltamexB strain . Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the mexB genes of a representative 46 were sequenced, and 19 unique single mutations were identified . While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops . By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from Escherichia coli, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it . Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition . Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction . As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.

Diagn Microbiol Infect Dis, 2004 Feb, 48(2), 131 - 5
Prevalence and characterization of metallo-beta-lactamases in clinical isolates of pseudomonas aeruginosa; Luzzaro F et al.; The prevalence and the type(s) of metallo-beta-lactamases (MBLs) produced by isolates of Pseudomonas aeruginosa were investigated . During 2001, 506 nonduplicate isolates were obtained from hospitalized patients . Eighty-two strains were selected because of resistance to carbapenems and/or ceftazidime . Screening for MBL production was performed in the latter isolates by the Etest MBL strips (AB Biodisk, Solna, Sweden) and by a broth microdilution method measuring minimum inhibitory concentrations (MICs) of imipenem alone and in the presence of metal-chelating agents (EDTA and o-phenanthroline) . Specific DNA probes were used to investigate the presence of genes coding for IMP- or VIM-type enzymes . Overall, four isolates of P . aeruginosa (obtained from independent patients) were found to carry a blaVIM gene . Polymerase chain reaction (PCR) experiments and DNA sequencing revealed that the VIM-2 determinant was present in three cases, whereas VIM-1 was detected in one isolate . Surveillance programs should be adopted to avoid the spread of these worrisome resistance genes.

J Cataract Refract Surg, 2004 Jan, 30(1), 257 - 8
Recurrent interface infiltration with hypopyon after astigmatic laser in situ keratomileusis on a penetrating corneal graft; Reinhard T et al.; A 56-year-old woman was referred with recurrent interface infiltration and hypopyon after astigmatic laser in situ keratomileusis (LASIK) on a corneal graft . Pseudomonas aeruginosa was isolated as the causative pathogen . Penetrating keratoplasty had been performed 2 years before refractive surgery . After the antibiotic medication was tapered, 3 recurrences of interface infiltration with hypopyon were observed . Penetrating rekeratoplasty was deemed appropriate . Histological examination of the explanted corneal graft revealed anterior stromal neutrophil infiltration . This case illustrates that microbial pathogens brought underneath the flap by LASIK can persist months later despite antimicrobial treatment.

J Cataract Refract Surg, 2004 Jan, 30(1), 195 - 9
Evaluation of preoperative and postoperative prophylactic regimens for prevention and treatment of diffuse lamellar keratitis; Holzer MP et al.; PURPOSE: To investigate preoperative and postoperative prophylactic treatment with different pharmacological agents before flap cutting and exposure to a diffuse lamellar keratitis (DLK) causative agent . SETTING: Magill Research Center for Vision Correction, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA . METHODS: The study comprised 48 eyes of 24 Dutch-belted rabbits . Three days before a corneal flap was cut and the corneal interface was exposed to Pseudomonas aeruginosa lipopolysaccharide endotoxin, a DLK causative agent, the eyes were randomly assigned to treatment with a mast-cell stabilizer, a nonsteroidal antiinflammatory drug (NSAID), or a corticosteroid or left without treatment as controls . The treatment was maintained throughout the 1-week follow-up . Slitlamp examinations and photographs were performed at 1, 3, 5, and 7 days; DLK was graded by a masked observer from 0 (no DLK) to IV . Corneal interface scrapings were performed in selected eyes on day 7 . RESULTS: At the end of the follow-up, 36 eyes were available for evaluation . At 1 week, 100% of the control eyes and the eyes treated with the mast-cell stabilizer developed DLK; in the NSAID-treated and corticosteroid-treated eyes, the DLK rate was 86% and 70%, respectively . At 1 day, the severity of DLK was significantly lower in eyes treated with the mast-cell stabilizer (0.44) and at 7 days, it was significantly lower in corticosteroid-treated eyes (0.3) than in the control group (1.5 and 1.4, respectively) (P<.05, Wilcoxon test) . Corneal interface scraping from an eye with grade III DLK showed numerous inflammatory cells . CONCLUSIONS: Preoperative and postoperative treatment with corticosteroids significantly reduced the severity of DLK compared to the untreated control eyes in this animal model . Treatment with a mast-cell stabilizer and an NSAID had less effect on the postoperative course of DLK.

J Korean Med Sci, 2004 Feb, 19(1), 62 - 8
Relationships between high-resolution computed tomography, lung function and bacteriology in stable bronchiectasis; Lee JH et al.; To determine the relationship between high-resolution computed tomography (HRCT) findings, lung function, and bacteriology in bronchiectasis, we conducted a retrospective study of 49 Korean patients with stable bronchiectasis . To quantify the extent and severity of bronchiectasis, we used a CT scoring system consisting of bronchial dilatation, bronchial wall thickening, the number of bronchiectatic segments, the number of bulla, and the number of emphysema segments . The presence of air-fluid levels and lung consolidation were also evaluated . The results of CT scoring, spirometry and sputum culture were analyzed . Patients with cystic bronchiectasis had higher CT score, more dilated lumen and lower forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV1), and FEV1/FVC than patients with cylindrical bronchiectasis . Patients with mixed ventilatory impairment had larger number of bronchiectatic segments than patients with obstructive ventilatory impairment . CT score and the number of bronchiectatic segments were significantly associated with FVC and FEV1, while CT score and the number of emphysema segments were significantly associated with FEV1/FVC . Twenty-one patients of 49 patients showed a positive sputum culture including 15 cases of Pseudomonas aeruginosa . The CT score was the most important predictor of lung function . The presence of air-fluid levels predicted bacterial colonization.

Lett Appl Microbiol, 2004, 38(3), 185 - 90
Liquid culture carbon, nitrogen and inorganic phosphate source regulate nematicidal activity by fluorescent pseudomonads in vitro; Siddiqui IA et al.; AIMS: The aim of the present investigation was to determine the influence of nutrients on the nematicidal activity by Pseudomonas aeruginosa strain IE-6S+ and Ps . fluorescens strain CHA0 in vitro . METHODS AND RESULTS: Culture filtrate of IE-6S+ and CHA0 obtained from chemically defined medium caused mortality of Meloidogyne javanica juveniles in vitro and that growth medium amended with various C, N or inorganic phosphate (Pi) sources markedly influenced nematicidal activity of the two bacteria . Glycerol (C source), propionate (fatty acid precursor) and L-lysine (N source) enhanced nematicidal activity while glucose (C), L-valine (N) and Pi substantially repressed nematicidal activity of the two bacteria . CONCLUSION: Liquid culture amendments with various C, N or Pi sources modulate the biosynthesis of nematicidal agents to a different extent in vitro . SIGNIFICANCE AND IMPACT OF THE STUDY: Developing bacterial strains more responsive to certain environmental signals can be exploited for increased secondary metabolite production in pharmaceutical fermentations and offers new avenues to improve biocontrol.

Rev Esp Quimioter, 2003 Dec, 16(4), 450 - 2
{Current status of Pseudomonas aeruginosa resistance to antibiotics}; Cobo Martinez F et al.; Pseudomonas aeruginosa is responsible for numerous infections, mainly in hospitals and in immunocompromised patients . This pathogen has a great ability to acquire resistance . Given the increase in prevalence of multiresistant isolates, a continuous analysis of the susceptibility of P . aeruginosa to antimicrobial drugs is required . We therefore evaluated all the P . aeruginosa isolates from clinical samples taken at our hospital in 2002, and included only one sample per patient . We studied the susceptibility of P . aeruginosa to 11 active antimicrobial drugs . Identification of the isolate and determination of susceptibility were carried out using an automated system (VITEK 2, bioMerieux) . Overall, the antibiotic with the highest in vitro activity was piperacillin-tazobactam . The percentage of multiresistant isolates (resistance to at least two groups of antibiotics) was 20.9% . In samples taken from the ICU, it showed high resistance to imipenem (20%) . P . aeruginosa isolated from abscesses was extremely resistant to gentamicin (41.6%) . The resistance of P . aeruginosa to antimicrobial drugs is inconsistent as it depends on the source of the isolates and the type of clinical samples . Consequently, individualized monitoring should be conducted.

Rev Esp Quimioter, 2003 Dec, 16(4), 421 - 7
{Trends in antimicrobial resistance of Pseudomonas aeruginosa, Escherichia coli and Bacteroides fragilis (1997-2001)}; Garcia-Rodriguez JA et al.; A study was conducted from 1997 to 2001 on the trends of the antibiotic resistance of Pseudomonas aeruginosa, Escherichia coli and Bacteroides fragilis in a Spanish multicenter study involving 26 hospitals . During the five years of the study the susceptibility by 81,779 strains of P . aeruginosa, 306,689 strains of E . coli and 2866 strains of B . fragilis to at least one antibiotic were studied . When the three microorganisms were considered together, meropenem (3.49%), piperacillin-tazobactam (5.54%) and imipenem (5.27%) were the antibiotics to which they showed the lowest resistance rate.

J Trauma, 2004 Feb, 56(2), 272 - 8
Burn injury and pulmonary sepsis: development of a clinically relevant model; Davis KA et al.; BACKGROUND: Despite improvements in the early resuscitation of the critically injured, mortality from multiple organ failure has remained stable, with the lung often the first organ to fail . Early intubation and mechanical ventilation predispose patients to the development of pneumonia and respiratory failure . Our objective was to establish a murine model of combined injury, consisting of burn/trauma and pulmonary sepsis with reproducible end-organ responses and mortality . METHODS: Male B6D2F1 mice were divided into four groups: burn/infection (BI), burn (B), infection (I), and sham (S) . Burned animals had a full-thickness 15% dorsal scald burn . BI and I groups were inoculated intratracheally with Pseudomonas aeruginosa (3-5 x 103 colony-forming units) . S and B animals received saline intratracheally . All animals were resuscitated with 2 mL of intraperitoneal saline . Mortality was recorded at 24, 48, and 72 hours . Bacterial sepsis was confirmed by tissue Gram's stain of the lungs and positive organ and blood cultures for Pseudomonas aeruginosa . Femoral bone marrow cells were collected at 72 hours from surviving animals . Clonogenic potential was assessed by response to macrophage (M) colony-stimulating factor (CSF) and granulocyte-macrophage (GM) CSF in a soft agar assay and the data were represented as colonies per femur . Isolated alveolar macrophages and whole lung tissue were assayed for levels of the inflammatory cytokines tumor necrosis factor-alpha and interleukin-6 . RESULTS: Mortality at 72 hours was 30% in BI, 12% in I, and <10% in B and S groups . Pneumonia was documented in all infected animals at 24 hours by Gram's stain and positive tissue cultures for Pseudomonas aeruginosa . Systemic sepsis as confirmed by blood, and remote organ cultures was seen in BI animals only . Significantly increased responsiveness to M-CSF stimulations was noted in all groups (BI, 8,291 +/- 1,402 colonies/femur; B, 6,357 +/- 806 colonies/femur; and I, 8,054 +/- 1,112 colonies/femur; p < 0.05) relative to sham (3,369 +/- 883 colonies/femur, p < 0.05) . Maximal responsiveness to GM-CSF stimulation was noted in the BI group (11,932 +/- 982 colonies/femur, p < 0.05), and similar GM responsiveness was noted in all other groups (B, 7,135 +/- 548 colonies/femur; I, 7,023 +/- 810 colonies/femur; and S, 6,829 +/- 1,439 colonies/femur) . Alveolar macrophage release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 increased in all animals, but the magnitude of increase was not proportional to the strength of the inciting stimulus . CONCLUSION: Although minimal perturbations were seen after burn or pulmonary infection alone, the combined insult of burn and pulmonary sepsis resulted in statistically significant hematopoietic changes with increased monocytopoiesis . Only the combined injury resulted in systemic sepsis and significantly increased mortality . We have developed a clinically relevant model of trauma and pulmonary sepsis that will allow further clarification of the inflammatory response after injury and infection.

Pediatr Infect Dis J, 2004 Feb, 23(2), 176 - 7
Pseudomonas aeruginosa septic shock secondary to "gripe water" ingestion; Sas D et al.; We report the case of a 9-month-old girl who presented in septic shock after ingestion of a contaminated herbal supplement commonly used to treat colic . Herbal supplements are widely used by well-meaning parents for many common conditions . Pediatricians should be aware that the variable manufacturing and packaging conditions of herbal supplements can lead to contamination with infectious agents.

Pediatr Infect Dis J, 2004 Feb, 23(2), 110 - 4
Use of DNA fingerprinting in decision making for considering closure of neonatal intensive care units because of Pseudomonas aeruginosa bloodstream infections; Schutze GE et al.; BACKGROUND: Bloodstream infections with Pseudomonas aeruginosa have been well-described in neonatal intensive care units (NICU) and have resulted in the temporary closure of some nurseries to new admissions . Nosocomial transmission of these infections has been verified by fingerprint analysis of the isolates . We utilized molecular fingerprinting to identify the source of bloodstream infections in an NICU and used this information to apply infection control measures that allowed the nursery to stay open and continue to accept referrals . METHODS: In June 1998 three premature infants transferred to our hospital (Hospital A) from Hospitals B and C had bloodstream infections with P . aeruginosa . Subsequently one additional neonate transferred from Hospital B was colonized with P . aeruginosa . Random amplification of polymorphic deoxyribonucleic acid (RAPD) was performed on the four isolates . All transfers from Hospital B were cultured, and surveillance programs were instituted in Hospitals A and B . Targeted infection control measures for all transfers were implemented . RESULTS: The four isolates were the same clone by RAPD . Investigation of the environment in Hospital A did not identify any source of the organism . Surveillance cultures on 49 neonates at Hospital A revealed only one patient colonized at an endotracheal tube . This patient was also a transfer from Hospital B . Results from Hospital B identified 4 of 40 (10%) neonates colonized . All isolates were clones identical with the bloodstream isolates from the neonates with bloodstream infections . Infection control measures for all babies transferred from Hospital B resulted in no new cases of P . aeruginosa bacteremia during the next 5 years . CONCLUSIONS: The use of molecular fingerprinting of isolates of P . aeruginosa allowed for a prompt and directed infection control plan to be implemented in Hospitals A and B . It also allowed the NICU in Hospital A to continue to accept referrals from other hospitals and to implement a targeted infection control plan for patients transferred from Hospital B.

Environ Microbiol, 2004 Mar, 6(3), 264 - 73
Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis; van Beilen JB et al.; The marine gamma-Proteobacterium Alcanivorax borkumensis is highly specialized in the assimilation of aliphatic hydrocarbons, and makes up a large part of the biomass in oil-polluted marine environments . In addition to the previously identified alkane hydroxylase AlkB1, a second alkane hydroxylase (AlkB2) showing 65% identity to the Pseudomonas aeruginosa AlkB2 alkane hydroxylase was identified . Unlike alkB1, alkB2 is not flanked by genes involved in alkane metabolism . Heterologous expression of the A . borkumensis AP1 alkB1 and alkB2 genes showed that they encode functional alkane hydroxylases with substrate ranges similar to those of their P . putida and P . aeruginosa homologues . The transcription initiation sites and levels of the alkB1, alkB2 and alkS mRNA transcripts were determined . Expression of both alkB1 and alkB2 was induced by alkanes, but transcripts corresponding to alkB1 were much more abundant than those of alkB2 . An inverted repeat similar to the binding site for the P . putida GPo1 transcriptional activator AlkS was present upstream of the promoters for alkB1 and alkB2, although that of alkB2 was less well conserved, and only the transcriptional fusion of promoter PalkB1 to the reporter gene lacZ efficiently responded to n-octane . Contrary to what has been found for the P . putida GPo1 alkane degradation pathway, expression of the A . borkumensis AP1 alkS gene was not induced by alkanes, and an AlkS binding site was not present upstream of the promoter for alkS . This indicates that, in spite of the clear similarities, the A . borkumensis alk-genes are regulated by a strategy different from that of the P . putida GPo1 alk genes.

Chest, 2004 Feb, 125(2), 607 - 16
Hospital mortality for patients with bacteremia due to Staphylococcus aureus or Pseudomonas aeruginosa; Osmon S et al.; STUDY OBJECTIVES: To evaluate the relationship between hospital mortality and bloodstream infections due to Staphylococcus aureus or Pseudomonas aeruginosa . DESIGN: Prospective cohort study . SETTING: A 1,400-bed, university-affiliated urban teaching hospital . PATIENTS: Between December 2001 and September 2002, 314 patients with bacteremia due to S aureus or P aeruginosa were prospectively evaluated . INTERVENTION: Prospective patient surveillance and data collection . RESULTS: Thirteen patients (4.1%) received inadequate initial antibiotic treatment . Fifty-four patients (17.2%) died during hospitalization . Hospital mortality was statistically greater for patients with bloodstream infections due to P aeruginosa (n = 49) compared to methicillin-sensitive S aureus (MSSA) {n = 117; 30.6% vs 16.2%, p = 0.036} and methicillin-resistant S aureus (MRSA) {n = 148; 30.6% vs 13.5%, p = 0.007} . Multiple logistic regression analysis identified the lack of response to initial medical treatment (adjusted odds ratio {AOR}, 2.69; 95% confidence interval {CI}, 1.83 to 3.94; p = 0.010) and endocarditis (AOR, 4.62; 95% CI, 2.45 to 8.73; p = 0.016) as independent determinants of hospital mortality . Patients with bloodstream infections due to P aeruginosa were statistically more likely to be nonresponders to early medical treatment compared to patients with MSSA (73.5% vs 11.1%, p < 0.001) and MRSA (73.5% vs 16.9%, p < 0.001) bloodstream infections . CONCLUSIONS: These data suggest that bloodstream infections due to P aeruginosa have a greater risk of hospital mortality compared to bloodstream infections due to S aureus despite adequate antibiotic treatment.

Acta Pharmacol Sin, 2004 Feb, 25(2), 239 - 45
PCR-based site-specific mutagenesis of peptide antibiotics FALL-39 and its biologic activities; Yang YX et al.; AIM: AIM: To construct PGEX-1lambdaT-FALL-39 expression vector and its mutant vector, and study the relationship of function and structure . METHODS: A cDNA encoding mature FALL-39 was cloned from SPCA-1 cell mRNA and the prokaryotic expression vector PGEX-1lambdaT-FALL-39 was constructed . Two kinds of polymerase chain reaction (PCR) for the site-direction mutagenesis were used to construct FALL-39 mutant expression vector, FALL-39-Lys-32 and FALL-39-Lys-24 . Minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration were used to assay the antibacterial activities of these peptides . Effects of different solution on the antibacterial activity of FALL-39 and FALL-39-Lys-32 were observed by CFU determination . The hemolytic effects of these peptides were also examined on human red blood cells . RESULTS: Two site-specific mutants FALL-39-Lys-32 and FALL-39-Lys24 were obtained by PCR-induced mutagenesis . In comparison with two-step PCR which required two pairs of primers, one step PCR which required one pair of primers is a simple and efficient method for the PCR based site-specific mutagenesis . Using the prokaryotic expression system, the E coli-based products of recombinant FALL39 and its mutant peptides were also obtained . The antibacterial assay showed that FALL-39-Lys-32 and FALL-39-Lys24 were more potential in the antibacterial activity against E coli ML35p and Pseudomonas aeruginosa ATCC27853 than that of FALL-39, and no increase in hemolysis was observed at the antibacterial concentrations . The antibacterial activity of FALL-39-Lys-32 against E coli was more potent than that of FALL-39 in NaCl-containing LB medium, while its activity was almost the same as FALL-39 in SO4(2-) containing Medium E . CONCLUSION: PCR-based mutagenesis is a useful model system for studying the structure and function relationship of antimicrobial peptides . Keeping a-helical conformation of FALL-39 and increasing net positive charge can increase the antibacterial activity of FALL-39 without increasing hemolysis at the antibacterial concentrations.

Proc Natl Acad Sci U S A, 2004 Feb 17, 101(7), 2135 - 9 Epub 2004 Feb 06.
The opportunistic pathogen Pseudomonas aeruginosa carries a secretable arachidonate 15-lipoxygenase; Vance RE et al.; In mammals, lipoxygenases play key roles in inflammation by initiating the transformation of arachidonic acid into potent bioactive lipid mediators such as leukotrienes and lipoxins . In general, most bacteria are believed to lack lipoxygenases and their polyunsaturated fatty acid substrates . It is therefore of interest that an ORF (PA1169) with high homology to eukaryotic lipoxygenases was discovered by analysis of the whole-genome sequence of the opportunistic bacterial pathogen Pseudomonas aeruginosa . Using TLC and liquid chromatography-UV-tandem mass spectrometry (LC-UV-MS-MS), we demonstrate that PA1169 encodes a bacterial lipoxygenase (LoxA) that converts arachidonic acid into 15-hydroxyeicosatetraenoic acid (15-HETE) . Although mammalian lipoxygenases are cytoplasmic enzymes, P . aeruginosa LoxA activity is secreted . Taken together, these results suggest a mechanism by which a pathogen-secreted lipoxygenase may modulate host defense and inflammation via alteration of the biosynthesis of local chemical mediators.

Appl Environ Microbiol, 2004 Feb, 70(2), 729 - 35
Effect of extracellular products of Pseudoalteromonas atlantica on the edible crab Cancer pagurus; Costa-Ramos C et al.; Previous studies have shown that injection of extracellular products (ECP) of Pseudoalteromononas atlantica isolated from shell disease-infected edible crabs (Cancer pagurus) into healthy crabs causes rapid death . In this study we examined the nature of the active lethal factor(s) in ECP . Injection of ECP into crabs caused a rapid decline in the total number of circulating hemocytes (blood cells), and the crabs died within 60 to 90 min . The individuals that died showed eyestalk retraction, limb paralysis, and lack of antennal sensitivity, suggesting that the active factor(s) targeted the nervous system . Histopathological investigations showed that affected crabs had large aggregates of hemocytes in the gills, and there was destruction of the tubules in the hepatopancreas . The active factor in ECP was not sensitive to heat treatment (100 degrees C for 30 min) and proteinase K digestion . As lipopolysaccharide (LPS) was a potential candidate for the lethal factor, it was purified from whole P . atlantica bacteria or ECP and subsequently injected into crabs . These crabs had all of the external symptoms observed previously with ECP, such as limb paralysis and eyestalk retraction, and they died within 90 min after challenge, although no significant decline in the number of circulating hemocytes was observed . Similarly, in vitro incubation of hemocytes with purified LPS (1 to 20 microg) from P . atlantica did not result in the clumping reaction observed with ECP but did result in a degranulation reaction and eventual cell lysis . Injection of crabs with Escherichia coli or Pseudomonas aeruginosa LPS (1 microg g of body weight(-1)) did not cause any of the characteristic symptoms observed following exposure to P . atlantica LPS . No mortality of crabs followed the injection of E . coli LPS, but P . aeruginosa LPS caused ca . 80% mortality at 2 h after injection . Overall, these results show that the main virulence factor of P . atlantica for edible crabs is LPS either alone or in combination with other heat-stable factors.

Prog Retin Eye Res, 2004 Jan, 23(1), 1 - 30
Corneal response to Pseudomonas aeruginosa infection; Hazlett LD; Pseudomonas aeruginosa (P . aeruginosa) is a common organism associated with bacterial keratitis, especially in those who use extended wear contact lenses . Recent advances in our understanding of host innate and adaptive immune responses to experimental infection have been made using a variety of animal models, including inbred murine models that are classed as resistant (cornea heals) vs . susceptible (cornea perforates) . Evidence has been provided that sustained IL-12-driven IFN-gamma production in dominant Th1 responder strains such as C57BL/6 (B6) contributes to corneal destruction and perforation, while IL-18-driven production of IFN-gamma in the absence of IL-12 is associated with bacterial killing and less corneal destruction in dominant Th2 responder strains such as BALB/c . The critical role of IL-1 and chemotactic cytokines such as MIP-2 in PMN recruitment and the critical role of this cell in the innate immune response to bacterial infection is reviewed . Regulation of PMN persistence is also discussed and evidence provided that persistence of PMN in B6 cornea is regulated by CD4+ T cells, while macrophages regulate PMN number in the cornea of BALB/c mice . The studies provide a better understanding of the inflammatory mechanisms that are operative in the cornea after P . aeruginosa challenge and are consistent with long-term goals of providing targets for alternative or adjunctive treatment for this disease . Future studies will be aimed at better defining the role of Toll receptors, neuropeptides (as unconventional modulators of the immune response) and exploitation of disease control by new techniques, such as RNA silencing.

Mol Microbiol, 2004 Feb, 51(4), 1193 - 203
Differences in two Pseudomonas aeruginosa cbb3 cytochrome oxidases; Comolli JC et al.; Bacterial cytochrome cbb3 oxidases are members of the haeme-copper oxidase superfamily that are important for energy conservation by a variety of proteobacteria under oxygen-limiting conditions . The opportunistic pathogen Pseudomonas aeruginosa is unusual in possessing two operons that each potentially encode a cbb3 oxidase (cbb3-1 or cbb3-2) . Our results demonstrate that, unlike typical enzymes of this class, the cbb3-1 oxidase has an important metabolic function at high oxygen tensions . In highly aerated cultures, cbb3-1 abundance and expression were greater than that of cbb3-2, and only loss of cbb3-1 influenced growth . Also, the activity of cbb3-1, not cbb3-2, inhibited expression of the alternative oxidase CioAB and thus influenced a signal transduction pathway much like that found in the alpha-proteobacterium Rhodobacter sphaeroides . Cbb3-2 appeared to play a more significant role under oxygen limitation by nature of its increased abundance and expression compared to highly aerated cultures, and the regulation of the cbb3-2 operon by the putative iron-sulphur protein Anr . These results indicate that each of the two P . aeruginosa cbb3 isoforms have assumed specialized energetic and regulatory roles.

Mol Microbiol, 2004 Feb, 51(4), 973 - 85
The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing; Schuster M et al.; In Escherichia coli and some other gamma-Proteobacteria, the alternative sigma factor RpoS functions as a regulator of the general stress response . The role of RpoS in Pseudomonas aeruginosa is not clear . Although P . aeruginosa RpoS contributes to the resistance to several environmental stresses, its role appears to be less pivotal than in E . coli . In P . aeruginosa, RpoS also regulates the production of several virulence factors and influences the expression of individual genes that are controlled by quorum sensing . Some quorum-controlled genes are induced by RpoS, whereas others are repressed . To gain insights about RpoS function in P . aeruginosa and to understand better the regulation of quorum-controlled genes, we used transcript profiling to define an RpoS regulon . We identified 772 genes regulated by RpoS in stationary but not in logarithmic growth phase (504 were induced and 268 were repressed), and we identified putative RpoS promoter sequence elements with similarity to the E . coli RpoS consensus in several of these genes . Many genes in the regulon, for example a set of chemotaxis genes, have assigned functions that are distinct from those in E . coli and are not obviously related to a stress response . Furthermore, RpoS affects the expression of more than 40% of all quorum-controlled genes identified in our previous transcriptome analysis . This highlights the significance of RpoS as a global factor that controls quorum-sensing gene expression at the onset of stationary phase . The transcription profiling results have allowed us to build a model that accommodates previous seemingly conflicting reports.

Gastroenterology, 2004 Feb, 126(2), 488 - 98
High-molecular-weight polyethylene glycol prevents lethal sepsis due to intestinal Pseudomonas aeruginosa; Wu L et al.; BACKGROUND & AIMS: During stress, erosion of protective intestinal mucus occurs in association with adherence to and disruption of the intestinal epithelial barrier by invading opportunistic microbial pathogens . The aims of this study were to test the ability of a high-molecular-weight polyethylene glycol compound, polyethylene glycol 15-20, to protect the intestinal epithelium against microbial invasion during stress . METHODS: The ability of polyethylene glycol 15-20 to protect the intestinal epithelium against the opportunistic pathogen Pseudomonas aeruginosa was tested in cultured Caco-2 cells . Bacterial virulence gene expression, bacterial adherence, and transepithelial electrical resistance were examined in response to apical inoculation of P . aeruginosa onto Caco-2 cells . Complementary in vivo studies were performed in a murine model of lethal sepsis due to intestinal P . aeruginosa in which surgical stress (30% hepatectomy) was combined with direct inoculation of P . aeruginosa into the cecum . RESULTS: High-molecular-weight polyethylene glycol (polyethylene glycol 15-20) conferred complete protection against the barrier-dysregulating effects of P . aeruginosa in Caco-2 cells . Intestinal application of polyethylene glycol 15-20 in stressed mice protected against the lethal effects of intestinal P . aeruginosa . Mechanisms of this effect seem to involve the ability of polyethylene glycol 15-20 to distance P . aeruginosa from the intestinal epithelium and render it completely insensate to key environmental stimuli that activate its virulence . CONCLUSIONS: High-molecular-weight polyethylene glycol has the potential to function as a surrogate mucin within the intestinal tract of a stressed host by inhibiting key interactive events between colonizing microbes and their epithelial cell targets.

Pathol Biol (Paris), 2004 Feb, 52(1), 33 - 8
{Recovery method of serotypable character in non serotypable pseudomonas aeruginosa strains}; Soler CP et al.; Serotyping is one of the most used techniques for typing Pseudomonas aeruginosa strains . During chronic infections, and especially in cystic fibrosis, the decrease of lipopolysaccharide production is responsible for difficulties in determining O antigens . The possibility of serotyping can be simply restored by using a primary culture broth containing amikacin (1/6 of the strain MIC for this antibiotic); this is due to the ability of this antibiotic to inhibit alginate production . This technique allowed us to determine the serotype of 108 non-serotypable strains of P . aeruginosa isolated in 14 different hospitals . Among these isolates, serotype O:1 and O:13, had a high prevalence; the origin is a deficiency in D-glucose and L-rhamnose, required for the synthesis of lipopolysaccharide . In contrast, these sugars are not present in lipopolysaccharide of O:12, and these strains are always serotypable . The main protein is Alg C; this bifunctional enzyme is required in the exopolysaccharide and lipopolysaccharide production, according stress conditions in the bacterial-cells' environment . Determination of the serotype, as Antibiogram, is essential for genotypic inquiries.

J Biol Inorg Chem, 2004 Apr, 9(3), 281 - 8 Epub 2004 Feb 03.
Methionine-121 coordination determines metal specificity in unfolded Pseudomonas aeruginosa azurin; Marks J et al.; Pseudomonas aeruginosa azurin binds copper so tightly that it remains bound even upon polypeptide unfolding . Copper can be substituted with zinc without change in protein structure, and also in this complex the metal remains bound upon protein unfolding . Previous work has shown that native-state copper ligands Cys112 and His117 are two of at least three metal ligands in the unfolded state . In this study we use isothermal titration calorimetry and spectroscopic methods to test if the native-state ligand Met121 remains a metal ligand upon unfolding . From studies on a point-mutated version of azurin (Met121Ala) and a set of model peptides spanning the copper-binding C-terminal part (including Cys112, His117 and Met121), we conclude that Met121 is a metal ligand in unfolded copper-azurin but not in the case of unfolded zinc-azurin . Combination of unfolding and metal-titration data allow for determination of copper (Cu(II) and Cu(I)) and zinc affinities for folded and unfolded azurin polypeptides, respectively.

Chembiochem, 2004 Feb 6, 5(2), 214 - 23
Learning from directed evolution: theoretical investigations into cooperative mutations in lipase enantioselectivity; Bocola M et al.; Molecular modeling with classical force-fields has been used to study the reactant complex and the tetrahedral intermediate in lipase-catalyzed ester hydrolysis in 20 enzyme/substrate combinations . The R and S enantiomers of alpha-methyldecanoic acid ester served as substrates for the wild-type lipase from Pseudomonas aeruginosa and nine selected mutants . After suitable preparation of initial structures from an available wild-type crystal structure, each system was subjected to 1 ns CHARMM force-field molecular dynamics simulations . The resulting geometric and energetic changes allow interpretation of some experimentally observed effects of mutations, particularly with regard to the "hot spots" at residues 155 and 162 . The replacement S155F enhances S enantiopreference through a steric relay involving Leu162 . The double mutation S53P + L162G improves S enantioselectivity by creating a new binding pocket for the S enantiomer with an additional stabilizing hydrogen bond to His83 . The simulations provide insight into remote and cooperative effects of mutations.

Am J Infect Control, 2004 Feb, 32(1), 27 - 30
Distinctive bacteria-binding property of cloth materials; Takashima M et al.; BACKGROUND: Nosocomial infections may be caused by pathogens that are transmitted from the hands or clothes of hospital personnel . Handwashing has been evaluated as effective against the spread of pathogens, but transmission through clothes has been little investigated . Evaluation of bacterial adherence to clothes is difficult because of the nonuniform amount of water absorbance by cloth . Therefore, we measured binding of bacteria to cloth fibers made of cotton, nylon, polyester, acrylic, or sheep's wool and tried to characterize bacterial binding to cloth . METHODS: We chose to study the opportunistic pathogens Staphylococcus aureus and Pseudomonas aeruginosa . Cloth fibers were incubated with bacterial suspensions in silicone-coated tubes . We evaluated the reduction of numbers of bacteria in solutions incubated with the fibers and calculated binding ratios of bacteria to the fibers . RESULTS: Polyester or acrylic fibers bound S aureus and P aeruginosa at high ratios (>80%), but cotton fibers bound them at low ratios (<10%) . Nylon fibers bound S aureus at low ratios, but P aeruginosa at intermediate ratios . CONCLUSION: The results suggested that polyester, acrylic, or wool clothes could be good carriers of S aureus and P aeruginosa and thus should be covered with cotton clothes to minimize the spread of the pathogens.

Am J Infect Control, 2004 Feb, 32(1), 12 - 6
Bacterial contamination of multiple-dose vials: a prevalence study; Mattner F et al.; BACKGROUND: Two patients died of a meningitis caused by Pseudomonas aeruginosa in a hospital in Germany in July 2001, their infections having been caused by a contaminated contrast media (iomeprol {Imeron}) used as a multiple-dose vial (MDV) over 8 days . Therefore, a prevalence study was performed to investigate the use and contamination of multiple-use vials in a tertiary hospital . METHODS: In a 1300-bed hospital on a specific day in November 2001, all used MDVs were collected by the infection control nurses . Information was recorded about the medication, labeling of vials, storing temperature, wards, and dates of opening . Each vial was also tested for sterility . RESULTS: Opened vials were to be found in all wards . Of the 227 vials available, 1 vial and 1 spike were contaminated with Staphylococcus epidermidis (contamination rate 0.9%; 95% CI, 0.3-2.1) . The opening dates were marked on only 114 (50%) MDVs, 15 (13%) of which had already expired . Only 44 (19%) MDVs had been stored in the refrigerator, whereas 109 MDVs contained medications without any preserving agent . CONCLUSION: Results revealed somewhat risky handling of MDVs . In light of a possible high risk in this hospital of about 1 contaminated MDV per day, and in view of many reported outbreaks induced by contaminated MDVs, the following infection control measures were encouraged: alcohol hand hygiene, the disinfection of gums, observance of the manufacturer's recommendations, appropriate storing temperatures, marking the opening time, and avoiding the multiple use of medications not containing preserving agents.

Shock, 2004 Feb, 21(2), 126 - 33
Inhibition of poly (ADP-ribose) polymerase attenuates acute lung injury in an ovine model of sepsis; Murakami K et al.; It is known that in various pathophysiological conditions, reactive oxidants cause DNA strand breakage and subsequent activation of the nuclear enzyme poly(ADP ribose) polymerase (PARP) . Activation of PARP results in cellular dysfunction . We hypothesized that pharmacological inhibition of PARP reduces the damage in the ovine model of acute lung injury (ALI) . After smoke inhalation, Pseudomonas aeruginosa (5 x 109 cfu/kg) was instilled into both lungs . All of the animals were mechanically ventilated with 100% O2 . The infusion of the PARP inhibitor (INO-1001, n = 6) began 1 h after the injury and thereafter through 24 h (3 mg bolus + 0.3 mg/kg/h, i.v.) . Control animals (n = 6) were treated with saline . Sham injury animals (n = 8) received sham smoke and were mechanically ventilated in the same fashion . One-half of those sham animals (n = 4) were given the same dose of INO-1001 . PaO2/FiO2 ratio at 24 h in saline and in the INO-1001-treated groups were 95 +/- 22 and 181 +/- 22, respectively (P < 0.05) . Peak airway pressure at 24 h in the saline- and INO-1001-treated groups was 32.6 +/- 3.0 and 24.4 +/- 2.2, respectively (P < 0.05) . Pulmonary shunt fraction was also significantly attenuated . INO-1001 treatment reduced pulmonary histological injury and attenuated poly (ADP-ribose) accumulation in the lung . In conclusion, inhibition of PARP improved the ALI after smoke inhalation and pneumonia . The results suggest that the activation of PARP plays a role in the pathophysiology of ALI in sheep.

J Antimicrob Chemother, 2004 Mar, 53(3), 451 - 6 Epub 2004 Jan 28.
Pseudomonas aeruginosa strains harbouring an unusual blaVIM-4 gene cassette isolated from hospitalized children in Poland (1998-2001); Patzer J et al.; OBJECTIVES: During 1997-2001, 151 isolates of imipenem-resistant Pseudomonas aeruginosa were obtained from clinical specimens taken from children hospitalized in Warsaw, Poland . These strains were investigated further to determine the mechanism of resistance . METHODS: The strains were analysed by a combination of genotyping and PCR-based strategies . RESULTS: Eleven of these strains were found to contain the metallo-beta-lactamase (M beta L) gene bla(VIM-4) . The first strain appeared in 1998, and P . aeruginosa strains harbouring this M beta L have become endemic in this hospital since then . All P . aeruginosa strains belonged to serotype O:6, and PFGE analysis revealed four different patterns and three sub-types . All 11 M beta L-producing strains contained an identical class 1 integron with the usual 5' and 3' conserved sequences . The integron included two resistance cassettes, aacA4 in the first position and the bla(VIM-4) cassette in the second position . The bla(VIM-4) gene included an unusual direct repeat of 169 bp of the 3' portion of the bla(VIM-4) gene . CONCLUSIONS: An unusual bla(VIM-4) M beta L has become endemic in P . aeruginosa isolates infecting Polish children hospitalized on surgical wards . The formation of this unusual bla(VIM-4) gene cassette could be explained by a mechanism involving deletion of a segment of an ancestral tandem repeat of bla(VIM-4) via slipped strand replication, mediated by a combination of polymerase and integrase.

Stat Med, 2004 Feb 15, 23(3), 493 - 508
A log-normal distribution model of the effect of bacteria and ear fenestration on hearing loss: a Bayesian approach; Gajewski BJ et al.; Chronic ear infection is a potentially life-threatening illness that medical doctors typically treat with ear surgery . Despite the success of this treatment, complications can occur due to bacteria infection . Surgeons believe that this infection causes the patient to have clinically significant hearing damage . In order to understand such complications, surgeons must quantify the effect of bacteria, their toxins and ear surgery on hearing loss . To this end, the other two authors of this paper performed two experiments on guinea pigs to measure hearing thresholds following a bacterial infection and surgery of the inner ear . The response variable in these experiments is hearing thresholds measured in decibels (dB) . The problem in analysing such experiments is that the hearing threshold observations often suffer from missing data and censoring mechanisms of various types . Additionally, the distribution of hearing thresholds has heavy tails and is peaked . In order to account for the above statistical issues, we present a Bayesian method with a location-shifted log-normal distribution . The method accounts for the uncertainty in the data collection mechanism and the parameters associated with a location-shifted log-normal distribution . We refer to one of the parameters as the "location-shift" parameter . The Bayesian approach provides a posterior distribution of the location-shift parameter that we compare with values estimated in previously published studies . The immediate goal of our proposed method was to quantify the effects of ear surgery and bacteria infection on hearing loss . Thus, we present the merits of the method in the form of a case study, and report posterior distributions of mean hearing loss, probability of clinically significant hearing loss and relative risk . The results show that surgeon 2, using the surgical procedure "oval window", poses a greater than 40 per cent chance of a 15dB hearing loss regardless of injection of bacteria or not . However, surgeon 1, using the surgical procedure "semicircular canal", does not pose a significantly greater than 40 per cent chance of a 15dB hearing loss unless there is a Pseudomonas aeruginosa-induced infection .

Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 337 - 9 Epub 2004 Jan 23.
Crystallization and preliminary X-ray diffraction analysis of an oxidized state of Ohr from Xylella fastidiosa; de Oliveira MA et al.; Xylella fastidiosa organic hydroperoxide-resistance protein (Ohr) is a dithiol-dependent peroxidase that is widely conserved in several pathogenic bacteria with high affinity for organic hydroperoxides . The protein was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 4000 as precipitant after treatment with organic peroxide (t-butyl hydroperoxide) . X-ray diffraction data were collected to a maximum resolution of 1.8 A using a synchrotron-radiation source . The crystal belongs to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 87.66, c = 160.28 A . The crystal structure was solved by molecular-replacement methods . The enzyme has a homodimeric quaternary structure similar to that observed for its homologue from Pseudomonas aeruginosa, but differs from the previous structure as the active-site residue Cys61 is oxidized . Structure refinement is in progress.

Biophys J, 2004 Feb, 86(2), 1149 - 59
Effects of cavity-forming mutations on the internal dynamics of azurin; Cioni P et al.; The effects of two single-point cavity-forming mutations, F110S and I7S, on the internal dynamics of azurin from Pseudomonas aeruginosa were probed by the phosphorescence emission of Trp-48, deeply buried in the compact hydrophobic core of the macromolecule . Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime (tau(0)) whereas more general effects on structural fluctuations were deduced from the phosphorescence acrylamide quenching rate constant (k(q)), which measures the diffusion of the solute through the protein fold . The results show a spectacular, 4-5 orders of magnitude, increase of k(q) emphasizing that large amplitude structural fluctuations permitting acrylamide migration to the protein core have been drastically enhanced in each azurin mutant . The large, 12-15 kcal/mol, decrease in the activation enthalpy associated to k(q) suggests that the rate enhancement is caused, rather than through a generalized increase of protein flexibility, by the elimination of an inner barrier to the diffusion process . According to tau(0) the chromophore environment is more fluid with I7S but strikingly more rigid with F110S, demonstrating that when internal cavities are formed local effects on the mobility at the mutation site are unpredictable . Both tau(0) and k(q) reveal a structure tightening role of bound Cd(2+) that correlates with the increase in stability from apo- to holo-azurin . While these alterations in internal dynamics of azurin do not seem to play a role on electron transfer through the central region, the enhanced migration of acrylamide emphasizes that cavities may be critical for the rapid diffusion of substrates to buried, solvent inaccessible sites of enzymes.

Zhonghua Er Ke Za Zhi, 2003 Jul, 41(7), 520 - 4
{Clinical analysis of 29 children with early infectious complications following hematopoietic stem cells transplantation}; Li Y et al.; OBJECTIVE: To study the clinical features and the incidence of early infectious complications following children hematopoietic stem cells transplantation (HSCT) . METHODS: The clinical data of 29 cases with early infectious complications following HSCT was retrospectively analyzed . RESULTS: The incidence of early infectious complications following HSCT in 31 children (including 22 cord blood transplantation and 9 peripheral blood stem cells transplantation) was 94% (29/31) . The first occurrence of the early infectious complications was at a median of 6 (0 - 22) days, the peak time of incidence was at a median of 4 - 7 days post transplantation . The duration of the first early infectious complications was at a median of 9 (3 - 20) days . The occurrence of the second early infectious complications was at a median of 19 (13 - 27) days . For all of the 29 children, when they developed early infectious complications their absolute neutrophil counts (ANC) were all > 0.5 x 10(9)/L . The most common infectious sites were the digestive tract (oral and gastro-intestinal mucositis) and then the respiratory tract . Gram negative blood infections were quite frequent and Pseudomonas aeruginosa was common in the oral-pharynx discharge cultures . Two children had Mycoplasma pneumonia infections and there were 4 incidences with fever but no definite infectious foci . The incidence and duration of early infectious complications following hematopoietic stem cells transplantation were associated with the duration of neutropenia . The source and the MNCs dose of the graft, the difference of conditioning regimen and GVHD prophylaxis method did not have a significant impact on the incidence and duration of early infectious complications . Antibiotic prophylaxis (including Tienam) could delay the occurrence of the early infections significantly . CONCLUSION: The incidence and duration of early infectious complications following hematopoietic stem cells transplantation were directly associated with the duration of neutropenia . Tienam regimen could postpone the early infections incidence and had effect of preventing the early infectious complications.

Klin Monatsbl Augenheilkd, 2004 Jan, 221(1), 52 - 5
{Nosocomial pseudomonas aeruginosa-associated keratitis in soft contact lens wearer}; Grunauer-Kloevekorn C et al.; BACKGROUND: Pseudomonas aeruginosa is the most common cause of bacterial-associated keratitis in soft contact lens wearers, due to wrong use of soft contact lenses . Problems are often severe corneal ulcers and even corneal perforations . We report on a soft contact lens wearer with credibly correct use of soft contact lenses and nosocomial Pseudomonas aeruginosa-associated keratitis . CASE REPORT: A 33-year old woman suffered from corneal ulcer and corneal infiltration with beginning endophthalmitis 2 days after having used of new soft contact lenses . After systemic and local antibiosis and penetrating keratoplasty we could stop endophthalmitis before reaching the vitreous and retina . RESULTS: Histological and microbiological examinations showed a corneal ulcer with severe corneal infection due to Pseudomonas aeruginosa with resistance to mezlocillin and intermediale resistance to gentamicin . After therapy a stable situation with visual acuity of 20/60 was attained . CONCLUSIONS: Previous reports on Pseudomonas aeruginosa-associated keratitis in soft contact lens wearers demonstrate corneal problems due to extended or overnight wear or unsuccessful contact lens cleaning . We present a case of nosocomial corneal infection after soft contact lens wearing and nosocomial infection because of contact with a partner working in an intensive-care unit . Hygienic rules should be strictly followed by patients and staff using soft or hard contact lenses for visual correction or for therapeutic reasons.

Ann Pharmacother, 2004 Feb, 38(2), 332 - 7 Epub 2003 Dec 19.
Single versus combined antibiotic therapy for gram-negative infections; Klibanov OM et al.; OBJECTIVE: To evaluate the available clinical data regarding single versus combination antimicrobial therapy for treatment of gram-negative infections, focusing on the more recent data in predominantly nonneutropenic hosts . In vitro and in vivo data regarding various antimicrobial combinations are also discussed . DATA SOURCES: Clinical trials, review articles, and meta-analyses were identified from a MEDLINE search (1960-July 2003) . Special attention was given to clinical outcome trials performed since 1989 . Search terms included gram-negative infections, drug synergism, Pseudomonas aeruginosa, monotherapy, combination therapy, carbapenems, beta-lactams, cefepime, aminoglycosides, and fluoroquinolones . DATA SYNTHESIS: Although most of the studies were not randomized, double-blind, or controlled, the most recent literature indicates that monotherapy with agents that are active against isolated organisms, including P . aeruginosa, may be appropriate for most patients . Efficacy outcomes, including mortality, did not significantly differ in most studies comparing single and combination therapies . Some trials suggest that combination therapy may be preferred in neutropenic patients and those with pseudomonal infections . CONCLUSIONS: Hospitalized patients with gram-negative infections are often treated with combination antimicrobial agents; however, some of the recently available data, although limited, suggest that administration of monotherapy is a feasible alternative in certain patient populations.

Antimicrob Agents Chemother, 2004 Feb, 48(2), 626 - 8
Class 1 integron containing metallo-beta-lactamase gene blaVIM-2 in Pseudomonas aeruginosa clinical strains isolated in Japan; Yatsuyanagi J et al.; Four bla(VIM-2) gene-harboring Pseudomonas aeruginosa strains were identified . These strains possessed a class 1 integron harboring ORF1, bla(VIM-2), and aacA4 gene cassettes . The transposon-mediated horizontal spread of the bla(VIM-2) gene among these strains was suggested, which increases the threat that the bla(VIM-2) gene will disseminate among diverse genera of bacteria.

Biomaterials, 2004 May, 25(11), 2139 - 51
Inhibition of bacterial adhesion on PVC endotracheal tubes by RF-oxygen glow discharge, sodium hydroxide and silver nitrate treatments; Balazs DJ et al.; Medical-grade poly(vinyl chloride) (PVC) was chemically modified to study how the incorporation of monovalent silver influences Pseudomonas aeruginosa adhesion and colonization . The modification investigated consisted of a radio frequency-oxygen (RF-O(2)) glow discharge pre-functionalization, followed by a two-step wet-treatment in sodium hydroxide and silver nitrate solutions . X-ray photoelectron spectroscopy (XPS) analysis and contact angle measurements were used to investigate the chemical nature and surface wettability of the films following each step of the modification . XPS analysis proved that the RF-O(2) plasma pre-functionalization of native PVC reproducibly increased the amount of functional groups representative of PVC additives, including ether/alcohol, esters and carboxyl groups . More specifically, we demonstrated that the O-C=O groups representative of the phthalic ester and zinc carboxylate additives identified for native PVC increased by two-fold following the RF-O(2) plasma pre-functionalization step . Although RF-O(2) pre-functionalization did not have an effect on the silver content of the NaOH/AgNO(3) treated substrates, such a modification was necessary for biomaterial products that did not have reproducible surfaces amongst production lots . XPS analysis also demonstrated that saponification with sodium hydroxide (NaOH) of esters, like those of the phthalic ester additives of PVC is a simple, irreversible method of hydrolysis, which produced sodium carboxylate and sodium phthalate salts . Exposure of native PVC to NaOH resulted in an increased surface hydrophilicity (from ca 90 degrees to ca 60 degrees ) due to dechlorination . XPS analysis following further incubation in silver nitrate demonstrated that silver ions can be trapped when the sodium of sodium carboxylate is replaced by silver after performing a second treatment with a monovalent silver-containing solution . The creation of silver salt on native PVC resulted in an ultra-hydrophobic (>120 degrees ) surface . The chemical modifications using NaOH and AgNO(3) wet treatments completely inhibited bacterial adhesion of four strains of P . aeruginosa to both native and oxygen-pre-functionalized PVC, and efficiently prevented colonization over longer periods (72 h) . Our results suggest that surface modifications that incorporate silver ions would be extremely effective at reducing bacterial colonization to medical devices.

Biomaterials, 2004 May, 25(12), 2247 - 63
Protein and bacterial fouling characteristics of peptide and antibody decorated surfaces of PEG-poly(acrylic acid) co-polymers; Wagner VE et al.; The potential for base poly(ethylene glycol) graft poly(acrylic acid) PEG-g-PA copolymers and surface-modified PEG-g-PA materials to inhibit random protein fouling and bacterial adhesion are investigated . PEG-g-PA co-polymers were synthesized that inhibited non-specific protein and cellular adhesion . PEG-g-PA co-polymers were then covalently modified with either cell adhesion peptides (YRGDS, YEILDV) or fragments of antibodies to monocyte/macrophage integrin receptors (Anti-VLA4, Anti-beta1, Anti-beta2, and Anti-CD64) known to enhance macrophage adhesion and, perhaps, modulate their activation . Materials produced in this work were characterized using: hydrophobicity by contact angle; angle-resolved X-ray Photoelectron Spectroscopy to confirm the presence of PEG in the bulk material and the surface; degree of hydration; differential scanning calorimetry; and thermal gravimetric analysis . To evaluate the non-fouling efficacy of the various modified surfaces, three proteins, human serum albumin, human fibronectin (Fraction I) and human immunoglobulin were 125I labeled . Samples of base PEG-g-PA and PEG-g-PA, modified with various peptides, were exposed to solutions containing either 2 or 200 microg/ml of one of the labeled proteins at 37 degrees C for 24 h . PEG-g-PA substrata modified with directly bound peptides exhibited protein adsorption that varied depending upon the surface bounded peptide . PEG-g-PA modified with peptides linked by linear PEG tethers reduced protein adsorption at 24 h by approximately 45% in comparison to PEG-g-PA . Peptides linked by way of StarPEO and StarlikePEO tethers further decreased protein adsorption in comparison to PEG-g-PA . The ability of peptide:PEOtethers to inhibit protein adsorption appeared to be a function of type and surface coverage of the PEO tether and not influenced by the amount or molecular structure the tethered peptide . Peptides directly coupled to the PEG-g-PA increased the amount of protein fouling relative to controls and there appeared to be some dependency of the amount of protein adsorption on which peptide was tethered . Two 14C-labeled pathogens, Staphylococcus epidermidis and Pseudomonas aeruginosa, were used to quantify the degree of bacterial adhesion using two types of laminar flow cell chambers; one that provided invasive sampling of the target substrata and one that provided non-invasive microscopic surveillance of adhering bacterial cells . Attachment of both species to PEG-g-PA and peptide-modified PEG-g-PA was reduced compared to the basic poly(acrylic acid) . Presence of peptides on the surface, whether directly bound or bound by the PEO tether did not influence adhesion of P . aeruginosa relative to controls . S . epidermidis adhesion rates increased slightly for those materials where peptides were directly bound to the surface but were reduced relative to base PEG-g-PA when peptides were bound by PEO tethers . All PEG-g-PA surfaces modified with fragments of monoclonal antibodies dramatically enhanced bacterial initial adhesion rates and maximum extent of attachment.

Middle East J Anesthesiol, 2003 Oct, 17(3), 371 - 8
Effects of different disinfectants on decontamination of laryngoscopes; Tekin I et al.; Guidelines for controlling possible contamination of laryngoscopes should be formulated with the benefit of relevant experimental data . In this study, the effects of five different disinfectants commonly used for the disinfection of laryngoscopes are evaluated . We formed 14 groups, with 10 blades in each . The first 7 groups were contaminated with hospital related meticillin resistant Staphylococcus aureus (MRSA), and the remaining 7 groups with hospital related multiple resistant Pseudomonas aeruginosa (PA) . For the first group of blades, no disinfection procedure was carried out and, were assumed as a control group . Blades in remaining groups were rested for 10 minutes in containers containing 70% alcohol (II), 1/100 dilution of cetrimide (III), 1/100 dilution of chlorhexidine (IV), 1/10 dilution of chlorhexidine (V), 1/10 dilution of povidone iodine (VI), and 1/100 dilution of ammonium chloride (VII) . Disinfectant used in a group was considered effective when growth was seen in 5 or less than 5 plates representing that group . All disenfectants tested were found effective on decontamination of laryngoscopes . Five different moderate level disinfectants, which are commonly used for the disinfection of laryngoscopes, have been found effective even on resistant hospital microorganisms like MRSA and P . aeruginosa . They may be the choices of the disinfectants, especially 1/10 dilution of chlorhexidine gluconate and 1/100 dilution of ammonium chloride.

Clin Exp Rheumatol, 2003 Nov-Dec, 21(6 Suppl 32), S95 - 100
Pseudomonas-induced lung damage in cystic fibrosis correlates to bactericidal-permeability increasing protein (BPI)-autoantibodies; Carlsson M et al.; OBJECTIVE: Lung damage is the most common cause of death in cystic fibrosis (CF) . It is induced by bacterial colonization and inflammatory activity perpetuates its course . Autoantibodies directed against BPI (bactericidal permeability increasing protein), called BPI-ANCA, have recently been associated with cystic fibrosis . Here we confirm this association and evaluate the relation between ANCA and total IgG level as they relate to bacterial colonization, pulmonary function, and musculoskeletal symptoms . METHODS: BPI-ANCA, MPO-ANCA, and PR3-ANCA were measured with ELISA in 46 adult patients with CF . Total IgG was determined by immunoturbidimetry . Results were correlated to bacterial colonization, lung function and musculoskeletal symptoms . RESULTS: BPI-ANCA was found in 33 patients . In the whole group, both BPI-ANCA and total IgG were inversely correlated to lung function, but in patients chronically colonized with Pseudomonas aeruginosa (P . aeruginosa), BPI-ANCA alone was correlated to lung damage (p = 0.01) . Median lung function, measured as forced expiratory volume in 1 second, in P . aeruginosa colonized patients with high levels of BPI-ANCA was 43% of the predicted value . In BPI-ANCA negative, the corresponding figure was 83% . In patients not colonized with P . aeruginosa, this relation was less evident . No correlation between ANCA and musculoskeletal symptoms was seen . CONCLUSION: P . aeruginosa induced lung damage in CF patients is associated with the presence of BPI-ANCA . P . aeruginosa colonized patients without BPI-ANCA have almost normal lung function . We suggest that BPI-ANCA discriminate P . aeruginosa colonized CF patients with severe lung damage from those whose disease is less destructive . Vasculitis like symptoms in CF are not ANCA associated.

Mol Genet Genomics, 2004 Mar, 271(2), 189 - 96 Epub 2004 Jan 23.
Cloning, expression and characterization of a lipase gene (lip3) from Pseudomonas aeruginosa LST-03; Ogino H et al.; A lipase gene (lip3) was cloned from the Pseudomonas aeruginosa strain LST-03 (which tolerates organic solvents) and expressed in Escherichia coli . The cloned sequence includes an ORF consisting of 945 nucleotides, encoding a protein of 315 amino acids (Lip3 lipase, 34.8 kDa) . The predicted Lip3 lipase belongs to the class of serine hydrolases; the catalytic triad consists of the residues Ser-137, Asp-258, and His-286 . The gene cloned in the present study does not encode the LST-03 lipase, a previously isolated solvent-stable lipase secreted by P . aeruginosa LST-03, because the N-terminal amino acid sequence of the Lip3 lipase differs from that of the LST-03 lipase . Although the effects of pH on the activity and stability of the Lip3 lipase, and the temperature optimum of the enzyme, were similar to those of the LST-03 lipase, the relative activity of the Lip3 lipase at lower temperatures (0-35 degrees C) was higher than that of the LST-03 lipase . In the absence of organic solvents, the half-life of the Lip3 lipase was similar to that of the LST-03 lipase . However, in the presence of most of the organic solvents tested in this study (the exceptions were ethylene glycol and glycerol), the stability of the Lip3 lipase was lower than that of the LST-03 lipase.

Intensive Care Med, 2004 Apr, 30(4), 693 - 701 Epub 2004 Jan 22.
Infectious and inflammatory dissemination are affected by ventilation strategy in rats with unilateral pneumonia; Schortgen F et al.; OBJECTIVE: To evaluate the effect of V(T) reduction and alveolar recruitment on systemic and contralateral dissemination of bacteria and inflammation during right-side pneumonia . DESIGN: Interventional animal study . SETTING . University hospital research laboratory . SUBJECTS: A total of 54 male Wistar rats . INTERVENTIONS: One day after right lung instillation of 1.4x10(7) Pseudomonas aeruginosa, rats were left unventilated or ventilated for 2 h at low V(T) (6 ml/kg) with different strategies of alveolar recruitment: no PEEP, 8 cm H(2)O PEEP, 8 cm H(2)O PEEP in a left lateral position, 3 cm H(2)O PEEP with partial liquid ventilation, or high V(T) (set such as end-inspiratory pressure was 30 cm H(2)O) without PEEP (ZEEP) . After ventilation the lungs, spleen and liver were cultivated for bacterial counts . Global bacterial dissemination was scored considering the percentage of positive spleen, liver and left lung cultures . TNF-alpha was assayed in plasma before and after mechanical ventilation . MEASUREMENTS AND RESULTS: All rats had right-side pneumonia with similar bacterial counts . All mechanical ventilation strategies, with the exception of low V(T)-PEEP 8, promoted contralateral lung dissemination . Overall bacterial dissemination was less in non-ventilated controls (22%) and low V(T)-PEEP 8 (22%) than in high V(T)-ZEEP (67%), low V(T)-PEEP 8 in left lateral position (59%) and low V(T)-ZEEP (56%) ( p<0.05) . Partial liquid ventilation prevented systemic bacterial translocation, but at the expense of contralateral bacterial seeding . Plasma TNF-alpha concentration increased significantly after mechanical ventilation with no PEEP at both high and low V(T) . CONCLUSIONS: Our results suggest that PEEP might reduce the risk of ventilation-induced bacterial and inflammatory mediator dissemination during pneumonia.

Biol Neonate, 2004, 85(4), 263 - 8 Epub 2004 Jan 21.
Ciprofloxacin treatment in newborns with multi-drug-resistant nosocomial Pseudomonas infections; Belet N et al.; OBJECTIVES: To evaluate the efficacy and acute side effects of ciprofloxacin treatment in newborns who developed nosocomial Pseudomonas aeruginosa infection . METHODS: Intravenous ciprofloxacin treatment was given to 30 newborns who developed nosocomial P . aeruginosa infection as proven by culture antibiogram results . Initial doses of 10 mg/kg/day were given and increased up to 40 mg/kg/day according to clinical response, laboratory and culture results . During therapy, complete white blood cell counts, urinalysis, liver and renal function tests were performed weekly . All patients were examined daily during treatment for possible symptoms of joint toxicity such as erythema and swelling . The patients were evaluated by general physical examination, with special attention to joints, 1 week after discharge . RESULTS: Two of the patients (6.6%) died due to pseudomonas infection, but the bacteria were successfully eradicated in 28 patients (93.4%) . Four patients died from other causes . No laboratory abnormality related to ciprofloxacin was observed during treatment . Swelling and hyperemia of the joints were not encountered during treatment and the 1-week period after discharge . Ciprofloxacin-resistant P . aeruginosa isolates were not grown during the study . CONCLUSION: Ciprofloxacin treatment is effective in life-threatening multi-drug-resistant P . aeruginosa infections .

Proc Natl Acad Sci U S A, 2004 Feb 3, 101(5), 1339 - 44 Epub 2004 Jan 22.
Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication; Deziel E et al.; Bacterial communities use "quorum sensing" (QS) to coordinate their population behavior through the action of extracellular signal molecules, such as the N-acyl-l-homoserine lactones (AHLs) . The versatile and ubiquitous opportunistic pathogen Pseudomonas aeruginosa is a well-studied model for AHL-mediated QS . This species also produces an intercellular signal distinct from AHLs, 3,4-dihydroxy-2-heptylquinoline (PQS), which belongs to a family of poorly characterized 4-hydroxy-2-alkylquinolines (HAQs) previously identified for their antimicrobial activity . Here we use liquid chromatography (LC)/MS, genetics, and whole-genome expression to investigate the structure, biosynthesis, regulation, and activity of HAQs . We show that the pqsA-E operon encodes enzymes that catalyze the biosynthesis of five distinct classes of HAQs, and establish the sequence of synthesis of these compounds, which include potent cytochrome inhibitors and antibiotics active against human commensal and pathogenic bacteria . We find that anthranilic acid, the product of the PhnAB synthase, is the primary precursor of HAQs and that the HAQ congener 4-hydroxy-2-heptylquinoline (HHQ) is the direct precursor of the PQS signaling molecule . Significantly, whereas phnAB and pqsA-E are positively regulated by the virulence-associated transcription factor MvfR, which is also required for the expression of several QS-regulated genes, the conversion of HHQ to PQS is instead controlled by LasR . Finally, our results reveal that HHQ is itself both released from, and taken up by, bacterial cells where it is converted into PQS, suggesting that it functions as a messenger molecule in a cell-to-cell communication pathway . HAQ signaling represents a potential target for the pharmacological intervention of P . aeruginosa-mediated infections.

Protein Sci, 2004 Feb, 13(2), 529 - 39
Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway; Webb NA et al.; d-Rhamnose is a rare 6-deoxy monosaccharide primarily found in the lipopolysaccharide of pathogenic bacteria, where it is involved in host-bacterium interactions and the establishment of infection . The biosynthesis of d-rhamnose proceeds through the conversion of GDP-d-mannose by GDP-d-mannose 4,6-dehydratase (GMD) to GDP-4-keto-6-deoxymannose, which is subsequently reduced to GDP-d-rhamnose by a reductase . We have determined the crystal structure of GMD from Pseudomonas aeruginosa in complex with NADPH and GDP . GMD belongs to the NDP-sugar modifying subfamily of the short-chain dehydrogenase/reductase (SDR) enzymes, all of which exhibit bidomain structures and a conserved catalytic triad (Tyr-XXX-Lys and Ser/Thr) . Although most members of this enzyme subfamily display homodimeric structures, this bacterial GMD forms a tetramer in the same fashion as the plant MUR1 from Arabidopsis thaliana . The cofactor binding sites are adjoined across the tetramer interface, which brings the adenosyl phosphate moieties of the adjacent NADPH molecules to within 7 A of each other . A short peptide segment (Arg35-Arg43) stretches into the neighboring monomer, making not only protein-protein interactions but also hydrogen bonding interactions with the neighboring cofactor . The interface hydrogen bonds made by the Arg35-Arg43 segment are generally conserved in GMD and MUR1, and the interacting residues are highly conserved among the sequences of bacterial and eukaryotic GMDs . Outside of the Arg35-Arg43 segment, residues involved in tetrameric contacts are also quite conserved across different species . These observations suggest that a tetramer is the preferred, and perhaps functionally relevant, oligomeric state for most bacterial and eukaryotic GMDs.

Microbes Infect, 2004 Jan, 6(1), 34 - 7
Effects of quorum-sensing on immunoglobulin G responses in a rat model of chronic lung infection with Pseudomonas aeruginosa; Wu H et al.; Levels of serum antibodies against Pseudomonas aeruginosa were observed for 106 days in a rat model of chronic lung infection . Significantly weaker responses of serum IgG and IgG1 and a lower ratio of IgG1/IgG2a were found in the rats infected with the quorum-signal-deficient mutant, PAO1 (rhlI, lasI), compared with the wild-type PAO1 . Four out of 15 rats infected with wild-type PAO1 contained bacteria in the lungs on day 106, whereas no bacteria were found in the mutant PAO1 group . The results indicate that quorum signals contribute to the persistence of the infection and influence the immune response.

Biomaterials, 2004 May, 25(10), 1947 - 57
Hemocompatibility of polyacrylonitrile dialysis membrane immobilized with chitosan and heparin conjugate; Lin WC et al.; Chitosan (CS)/heparin (HEP) polyelectrolyte complex (PEC) was covalently immobilized onto the surface of polyacrylonitrile (PAN) membrane . The effect of surface modification on the protein adsorption and platelet adhesion, metabolites permeation and anticoagulation activity of the resulting membrane was investigated . Surface characterization such as water contact angle, and X-ray photoelectron spectroscope were performed . The immobilization of PEC caused the water contact angle to reduce, thereby indicating the increase in the hydrophilicity . Protein adsorption, platelet adhesion, and thrombus formation were all reduced by the immobilization of HEP . Anticoagulant activity was evaluated with activated partial thrombin time (APTT), prothrombin time (PT), fibrinogen time, and thrombin time (TT) . The results revealed that PEC-immobilizing membrane can improve antithrombogenicity of PAN membrane . In addition, the PEC-immobilized membranes can suppress the proliferation of Pseudomonas aeruginosa . In vitro cytotoxicity test showed leachable substance released was below cytotoxic level . The pure water permeability results show little variation due to PEC-immobilization . Thus PEC-immobilization can endow the PAN membrane hemocompatibility and antibacterial activity while retaining the original permeability.

Clin Exp Immunol, 2004 Feb, 135(2), 240 - 6
Stimulation of innate immunity by susceptible and multidrug-resistant Pseudomonas aeruginosa: an in vitro and in vivo study; Giamarellos-Bourboulis EJ et al.; In attempt to investigate the stimulatory effect of Pseudomonas aeruginosa on innate immunity and to correlate it to its level of resistance to antimicrobials, 20 isolates were applied; 8 isolates were susceptible and 12 multidrug-resistant . Genetic diversity was defined by PFGE . Human monocytes of two healthy volunteers were in vitro stimulated by the isolates for the production of pro-inflammatory (TNF-alpha, IL-1beta, IL-6, IL-8 and IL-12) and anti-inflammatory cytokines (IL-10), of malondialdehyde and of procalcitonin . Cytokines were estimated by EIA, malondialdehyde by the thiobarbiturate assay and procalcitonin by an immunochemiluminometric assay . Survival of 48 Wistar rats was recorded after induction of sepsis by the intraperitoneal injection of three susceptible and three multidrug-resistant isolates . To test whether comparative effect of the latter isolates on survival correlates with any difference of monocyte-mediated release of pro-inflammatory mediators, monocytes of two rats were in vitro stimulated for the production of TNF-alpha and of malondialdehyde . In vitro stimulation of human monocytes by the susceptible isolates elicited elevated production of malondiadeheyde, of IL-1beta and of IL-6 compared to stimulation by multidrug-resistant isolates . Similar differences were found for TNF-alpha and IL-8, but they were not statistically significant . Production of IL-10 and IL-12 was not detected after stimulation with any isolate . Levels of procalcitonin were similar after induction with either susceptible or multidrug-resistant isolates . Mean survival of animals was 7.56, 21.80 and 55.20 h, respectively, after challenge by the susceptible isolates and 28.89, 61.8 and more than 120 h, respectively, after challenge by the multidrug-resistant isolates . Differences of survival were accompanied by greater rodent monocyte-release of TNF-alpha and malondialdehyde after stimulation by the susceptible isolates compared to multidrug-resistant ones . It is concluded that considerable differences are encountered on the stimulation of human monocytes by susceptible and resistant isolates of Pseudomonas aeruginosa . These results correlate with in vivo evidence and might influence decision on therapeutics.

Br J Ophthalmol, 2004 Feb, 88(2), 165 - 6
Distribution and shifting trends of bacterial keratitis in north China (1989-98); Sun X et al.; AIMS: To study the distribution and shifting trends of bacterial keratitis . METHODS: The data of 2220 corneal isolates from 1 January 1989 to 31 December 1998 were reviewed retrospectively . RESULTS: Positive culture was recovered in 490 isolates . Gram positive cocci and Gram negative bacilli represented 51% and 39.4%, respectively . Pseudomonas aeruginosa was the most common pathogen (32.2%) . A gradual increase in the percentage of Gram positive cocci coupled with a decrease of Gram negative bacilli . CONCLUSION: Pseudomonas aeruginosa and coaculase negative Staphylococcus were the most common pathogens in bacterial keratitis in north China.

J Immunol, 2004 Feb 1, 172(3), 1801 - 8
p47phox deficiency impairs NF-kappa B activation and host defense in Pseudomonas pneumonia; Sadikot RT et al.; We examined the role of redox signaling generated by NADPH oxidase in activation of NF-kappaB and host defense against Pseudomonas aeruginosa pneumonia . Using mice with an NF-kappaB-driven luciferase reporter construct (HIV-LTR/luciferase (HLL)), we found that intratracheal administration of P . aeruginosa resulted in a dose-dependent neutrophilic influx and activation of NF-kappaB . To determine the effects of reactive oxygen species generated by the NADPH oxidase system on activation of NF-kappaB, we crossbred mice deficient in p47(phox) with NF-kappaB reporter mice (p47(phox-/-)HLL) . These p47(phox-/-)HLL mice were unable to activate NF-kappaB to the same degree as HLL mice with intact NADPH oxidase following P . aeruginosa infection . In addition, lung TNF-alpha levels were significantly lower in p47(phox-/-)HLL mice compared with HLL mice . Bacterial clearance was impaired in p47(phox-/-)HLL mice . In vitro studies using bone marrow-derived macrophages showed that Toll-like receptor 4 was necessary for NF-kappaB activation following treatment with P . aeruginosa . Additional studies with macrophages from p47(phox-/-) mice confirmed that redox signaling was necessary for maximal Toll-like receptor 4-dependent NF-kappaB activation in this model . These data indicate that the NADPH oxidase-dependent respiratory burst stimulated by Pseudomonas infection contributes to host defense by modulating redox-dependent signaling through the NF-kappaB pathway.

FEMS Microbiol Lett, 2004 Jan 15, 230(1), 143 - 6
Detection of an IS21 insertion sequence in the mexR gene of Pseudomonas aeruginosa increasing beta-lactam resistance; Boutoille D et al.; To understand the regulation of the MexAB OprM efflux system in a clinical strain of Pseudomonas aeruginosa presenting a decreased susceptibility to ticarcillin and aztreonam, the mexR repressor gene was amplified by polymerase chain reaction (PCR) and was shown to be disrupted by an insertion sequence of more than 2 kb, with characteristic direct and inverted repeat sequences . Sequencing revealed a 2131-bp IS21 insertion sequence . A reverse transcription PCR method was used to quantify mexA transcripts and showed an increased transcription rate of mexA in this strain, compared with a PAO1 control strain . The nalB phenotype in P . aeruginosa may be due to point mutations, but also to the presence of an insertion sequence in the mexR regulator gene.

FEMS Microbiol Lett, 2004 Jan 15, 230(1), 27 - 34
Dueling quorum sensing systems in Pseudomonas aeruginosa control the production of the Pseudomonas quinolone signal (PQS); McGrath S et al.; The opportunistic human pathogen Pseudomonas aeruginosa regulates the production of numerous virulence factors via the action of two separate but coordinated quorum sensing systems, las and rhl . These systems control the transcription of genes in response to population density through the intercellular signals N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C(12)-HSL) and N-(butanoyl)-L-homoserine lactone (C(4)-HSL) . A third P . aeruginosa signal, 2-heptyl-3-hydroxy-4-quinolone {Pseudomonas quinolone signal (PQS)}, also plays a significant role in the transcription of multiple P . aeruginosa virulence genes . PQS is intertwined in the P . aeruginosa quorum sensing hierarchy with its production and bioactivity requiring the las and rhl quorum sensing systems, respectively . This report presents a preliminary transcriptional analysis of pqsA, the first gene of the recently discovered PQS biosynthetic gene cluster . We show that pqsA transcription required pqsR, a transcriptional activator protein encoded within the PQS biosynthetic gene cluster . It was also found that the transcription of pqsA and subsequent production of PQS was induced by the las quorum sensing system and repressed by the rhl quorum sensing system . In addition, PQS production was dependent on the ratio of 3-oxo-C(12)-HSL to C(4)-HSL, suggesting a regulatory balance between quorum sensing systems . These data are an important early step toward understanding the regulation of PQS synthesis and the role of PQS in P . aeruginosa intercellular signaling.

J Endotoxin Res, 2003, 9(6), 395 - 400
Pseudomonas aeruginosa lipid A diversity and its recognition by Toll-like receptor 4; Ernst RK et al.; Lipid A is the pro-inflammatory component of bacterial lipopolysaccharide, the major surface component of Gram-negative bacteria . Gram-negative bacteria alter the structure of lipid A in response to specific environmental conditions including those found upon colonization of a host . The opportunistic pathogen Pseudomonas aeruginosa synthesizes a unique hexa-acylated lipid A containing palmitate and aminoarabinose during adaptation to the cystic fibrosis airway . Different lipid A species are observed in P . aeruginosa isolated from non-cystic fibrosis associated infections . Here we report that P . aeruginosa isolates from the airway of a cystic fibrosis patient with severe pulmonary disease synthesized a novel hepta-acylated lipid A . Cystic fibrosis-specific P . aeruginosa lipid A modifications result in resistance to host antimicrobial peptides and increased recognition by human Toll-like receptor 4 (TLR4) . Using P . aeruginosa lipid A with different levels of acylation, we identified a 222 amino acid region in the extracellular portion of human TLR4 that is required for the differential recognition of cystic fibrosis-specific lipid A . P . aeruginosa adaptation to the human airway may, therefore, play a fundamental role in the progressive lung damage associated with cystic fibrosis.

Biochem J, 2004 May 1, 379(Pt 3), 563 - 72
Elucidation of eukaryotic elongation factor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exotoxin A; Yates SP et al.; Pseudomonas aeruginosa produces the virulence factor, ETA (exotoxin A), which catalyses an ADP-ribosyltransferase reaction of its target protein, eEF2 (eukaryotic elongation factor-2) . Currently, this protein-protein interaction is poorly characterized and this study was aimed at identifying the contact sites between eEF2 and the catalytic domain of ETA (PE24H, an ETA from P . aeruginosa, a 24 kDa C-terminal fragment containing a His6 tag) . Single-cysteine residues were introduced into the toxin at 21 defined surface-exposed sites and labelled with the fluorophore, IAEDANS {5-(2-iodoacetylaminoethylamino)-1-napthalenesulphonic acid} . Fluorescence quenching studies using acrylamide, and fluorescence lifetime and wavelength emission maxima analyses were conducted in the presence and absence of eEF2 . Large changes in the microenvironment of the AEDANS {5-(2-aminoethylamino)-1-naphthalenesulphonic acid} probe after eEF2 binding were not observed as dictated by both fluorescence lifetime and wavelength emission maxima values . This supported the proposed minimal contact model, which suggests that only small, discrete contacts occur between these proteins . As dictated by the bimolecular quenching constant (k(q)) for acrylamide, binding of eEF2 with toxin caused the greatest change in acrylamide accessibility (>50%) when the fluorescence label was near the active site or was located within a known catalytic loop . All mutant proteins showed a decrease in accessibility to acrylamide once eEF2 bound, although the relative change varied for each labelled protein . From these data, a low-resolution model of the toxin-eEF2 complex was constructed based on the minimal contact model with the intention of enhancing our knowledge on the mode of inactivation of the ribosome translocase by the Pseudomonas toxin.

J Surg Res, 2004 Jan, 116(1), 137 - 44
Cytokine induction by the P . aeruginosa quorum sensing system during thermal injury; Rumbaugh KP et al.; INTRODUCTION: Pseudomonas aeruginosa causes serious infections in severely burned patients due to its ability to produce numerous virulence factors . The production of most of these factors is controlled by the cell-to-cell communication system called quorum sensing (QS) . We have recently shown that several proinflammatory and hematopoietic cytokines are produced during infection of the burn wound with P . aeruginosa strain PAO1 . Most of these cytokines were not produced during either thermal injury or P . aeruginosa infection alone . MATERIALS AND METHODS AND RESULTS: In this study, we tried to determine if the QS systems play a role in the production of cytokines during P . aeruginosa infection of burn wounds . This was accomplished using the murine model of thermal injury, the P . aeruginosa strain PAO1 and its QS defective mutant (PAO-JP2), and the Multi-probe RNase protection assay . The mRNA for TNF-alpha, IL-6, TGF-beta, and G-CSF was detected within the skin of PAO1 infected/thermally injured mice . In contrast, the expression of these cytokines was not detected in PAO-JP2 infected/thermally injured mice . In comparison with the parent strain, PAO-JP2 was not defective either in its growth or in its spread within the thermally injured skin . A complementation experiment, using a plasmid that carries the intact QS gene, was conducted to confirm these results . In the presence of the complementing plasmid, PAO-JP2 produced the mRNA for the above cytokines . CONCLUSIONS: These results suggest that: 1) the QS system is involved in the induction of cytokine expression during P . aeruginosa infection of burn wounds; and 2) this effect may be caused by either a component of the QS system or a QS-controlled virulence factor.

Free Radic Biol Med, 2004 Jan 1, 36(1), 90 - 100
Direct oxidation of 2',7'-dichlorodihydrofluorescein by pyocyanin and other redox-active compounds independent of reactive oxygen species production; O'Malley YQ et al.; Formation of dichlorofluorescein (DCF), the fluorescent oxidation product of 2',7'-dichlorodihydrofluorescein (DCFH2), in cells loaded with the latter compound is often used to detect ROS formation . We previously found that exposure of DCFH2-loaded A549 cells to the Pseudomonas aeruginosa secretory product pyocyanin results in DCF formation, consistent with ROS production . However, since pyocyanin directly accepts electrons from NAD(P)H, we hypothesized that pyocyanin might directly oxidize DCFH2 to DCF without an ROS intermediate . Incubation of DCFH2 with pyocyanin rapidly resulted in DCF formation, the rate of which was proportional to the {pyocyanin} and was not inhibited by SOD or catalase . Phenazine methosulfate, a pyocyanin analog, was more effective than pyocyanin in generating DCF . Mitoxantrone and ametantrone also produced DCF . However, menadione, paraquat, plumbagin, streptonigrin, doxorubicin, daunorubicin, and 5-iminodaunorubicin did not . Pyocyanin, phenazine methosulfate, mitoxantrone, and ametantrone also oxidized dihydrofluorescein and 5- (and 6-) -carboxy-2',7'-dichlorodihydrofluorescein, whereas dihydrorhodamine was oxidized only by pyocyanin or phenazine methosulfate . Under aerobic conditions, the interaction of DCFH2 with pyocyanin or phenazine methosulfate (but not mitoxantrone or ametantrone) produced superoxide, as detected by spin trapping . Direct oxidation of the fluorescent probes needs to be controlled for when employing these compounds to assess ROS formation by biological systems exposed to redox active compounds.

Pediatr Pulmonol, 2004 Feb, 37(2), 104 - 10
Reduction in prevalence of chronic Pseudomonas aeruginosa infection at a regional pediatric cystic fibrosis center; Lee TW et al.; Various management strategies were introduced at the Leeds Regional Cystic Fibrosis (CF) Unit in an attempt to reduce the prevalence of chronic Pseudomonas aeruginosa respiratory infection, previously thought to be inevitable in most children with CF . These included neonatal screening (1975), regular microbiological monitoring (1975), early antibiotic treatment of first isolations of P . aeruginosa (1985), intensive intravenous antibiotic treatment where nebulized antibiotics failed to eradicate P . aeruginosa (1988), and separate clinics for patients chronically infected with P . aeruginosa and uninfected patients (1991) . The aim of this study was to assess the impact of these interventions . All 232 patients receiving full-time care at the Leeds Paediatric CF Centre during the period January 1990-December 2000 were categorized into four groups: never grown P . aeruginosa; free of P . aeruginosa for at least 1 year; intermittent grower of P . aeruginosa with </=50% of months with samples positive for P . aeruginosa over the previous 12 months; and chronic P . aeruginosa infection with >50% of months with samples positive for P . aeruginosa over the previous 12 months . The yearly prevalence of patients having chronic P . aeruginosa infection fell significantly during the study, from 24.5% in 1990 to 18.1% in 2000 (P < 0.05), despite an increase in mean age of patients from 7.73 to 9.42 years . The number of patients aged less than 11 years who had chronic P . aeruginosa infection fell from 23.8% in January 1990 to only 4.3% by December 2000 . The annual incidence and mean age of first acquisition of P . aeruginosa did not alter significantly . In conclusion, antipseudomonal management strategies were associated with both reduced prevalence, and an increase in the mean age of onset of chronic P . aeruginosa infection .

J Antimicrob Chemother, 2004 Feb, 53(2), 297 - 304 Epub 2004 Jan 16.
Pharmacodynamic study of beta-lactams alone and in combination with beta-lactamase inhibitors against Pseudomonas aeruginosa possessing an inducible beta-lactamase; Li C et al.; OBJECTIVES: The antimicrobial efficacies of beta-lactams alone and in combination with beta-lactamase inhibitors were investigated by applying a rabbit tissue cage model against a strain of Pseudomonas aeruginosa with an inducible AmpC (iAmpC) beta-lactamase . METHODS: Two sterilized golf Wiffle balls were surgically implanted in the rabbit dorsal cervical area . After 4 weeks, Wiffle balls had filled with tissue cage fluid (TCF), in which 2 mL of 10(6) cfu/mL of the test isolate were inoculated . To achieve the same T > MIC as in humans, 400 mg/kg of the beta-lactams alone and in combination was administered twice a day via subcutaneous injection . The dosing regimens were as follows: piperacillin alone, 4 g piperacillin/0.5 g tazobactam; ticarcillin alone, 3 g ticarcillin/0.1 g clavulanate; and 3 g ticarcillin/ 0.3 g clavulanate . RESULTS: The changes in bacterial counts (log cfu/mL) after the 3 day treatments were as follows: 1.03 +/- 0.97 (control), -1.31 +/- 0.61 (piperacillin), -2.81 +/- 0.53 (4 g piperacillin/0.5 g tazobactam), -1.61 +/- 0.68 (ticarcillin), -3.42 +/- 0.75 (3 g ticarcillin/0.1 g clavulanate) and -1.65 +/- 1.47 log cfu/mL (3 g ticarcillin/0.3 g clavulanate) . AmpC induction by high-dose clavulanate was observed in rabbit TCF, and was confirmed by the in vitro induction study . CONCLUSIONS: The study indicated that tazobactam significantly enhanced the antibacterial activity of piperacillin against iAmpC P . aeruginosa; clavulanate had synergy with the antibacterial activity of ticarcillin at low concentration, but had no effect on ticarcillin at high concentration due to AmpC induction by clavulanate.

Cancer Res, 2004 Jan 1, 64(1), 400 - 5
Enhancement of DNA vaccine potency by coadministration of a tumor antigen gene and DNA encoding serine protease inhibitor-6; Kim TW et al.; Serine protease inhibitor 6 (SPI-6), also called Serpinb9, inhibits granzyme B and thus may provide a method for delaying apoptotic cell death in dendritic cells . We have previously enhanced DNA vaccine potency by targeting antigen to MHC antigen presentation pathways, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, domain II of Pseudomonas aeruginosa exotoxin A, or the sorting signal of the lysosome-associated membrane protein type 1 . In this study, we explored intradermal coadministration of DNA encoding SPI-6 with DNA constructs encoding human papillomavirus type 16 E7 linked to these intracellular targeting molecules for its ability to generate E7-specific CD8+ T-cell immune responses and E7-specific antitumor effects . This combination of strategies resulted in significantly increased E7-specific CD8+ T-cell and CD4+ Th1-cell responses, enhanced tumor treatment ability, and stronger tumor protection when compared with vaccination without SPI-6 . Among these targeting strategies tested, mice vaccinated with Sig/E7/lysosome-associated membrane protein type 1 mixed with SPI-6 showed the greatest fold increase in E7-specific CD8+ T cells ( approximately 5-fold) . Vaccination with a nonfunctional mutant of SPI-6 did not result in immune enhancement, indicating that enhancement was dependent on the antiapoptotic function of SPI-6 . Our results suggest that DNA vaccines combining strategies that enhance MHC class I and II antigen processing with SPI-6 have potential clinical implications for control of viral infection and neoplasia.

J Inorg Biochem, 2004 Feb, 98(2), 276 - 86
Two azurins with unusual redox and spectroscopic properties isolated from the Pseudomonas chlororaphis strains DSM 50083T and DSM 50135; Pinho D et al.; Two azurins (Az624 and Az626) were isolated from the soluble extract of two strains of Pseudomonas chlororaphis, DSM 50083(T) and DSM 50135, respectively, grown under microaerobic conditions with nitrate as final electron acceptor . The azurins, purified to electrophoretic homogeneity in three chromatographic steps, exhibit several peculiar properties . They have high reduction potentials and lower pI than most azurins described in the literature . As previously observed for Pseudomonas aeruginosa azurin, their reduction potentials are pH-dependent, but the pK values of their oxidized forms are lower, which suggests that deeper structural changes are associated with the oxidation process of these novel azurins . A hitherto undescribed pH-dependence of the diffusion coefficient was observed in Az624, that could be caused either by conformational changes, or by the formation of supramolecular aggregates associated with a protonation process . Both azurins exhibit axial X-band electron paramagnetic resonance spectra in frozen solution showing a typical hyperfine with the copper nucleus (I=3/2) and a well-resolved superhyperfine structure with two equivalent 14N nucleus (I=1), which is not usually observed for azurins from other species.

Transplantation, 2004 Jan 15, 77(1), 134 - 6
Effects of sinus surgery in patients with cystic fibrosis after lung transplantation: a 10-year experience; Holzmann D et al.; Chronic infectious rhinosinusitis with Pseudomonas aeruginosa is common in cystic fibrosis and may result in allograft infection after lung transplantation . Sinus surgery followed by nasal care may reduce these adverse effects . Sinus surgery was performed in 37 patients with cystic fibrosis after transplantation . Bacteriology of sinus aspirates (n=771) and bronchoalveolar lavage (BAL) (n=256) was correlated with clinical data . Sinus surgery was successful in 54% and partially successful in 27% of patients . A significant correlation between negative sinus aspirates and negative BAL and between positive sinus aspirates and positive BAL (P<0.0001) was found . Successful sinus management led to a lower incidence of tracheobronchitis and pneumonia (P=0.009) and a trend toward a lower incidence of bronchiolitis obliterans syndrome (P=0.23) . Sinus surgery followed by daily nasal douching may control posttransplant lower airway colonization and infection . In the long term, this concept may lead to less bronchiolitis obliterans syndrome by decreasing bronchiolar inflammation.

J Appl Microbiol, 2004, 96(2), 244 - 53
The synergistic effect of EDTA/antimicrobial combinations on Pseudomonas aeruginosa; Lambert RJ et al.; AIMS: To demonstrate that the nonlinear concentration-dependent inhibition of Pseudomonas aeruginosa to EDTA can be used to successfully model and predict the potentiation of antimicrobials by EDTA . METHODS AND RESULTS: A model used successfully to describe the concentration-dependent inhibition of bacterial growth caused by many antimicrobials was unable to describe the inhibition of P . aeruginosa by EDTA . Examination of the inhibition profiles for EDTA against P . aeruginosa revealed a biphasic inhibitory pattern suggesting different mechanisms of action at different concentrations . A modelled, two-stage inhibitory process was shown to fit the observations . This model was then used to examine the effect of combining EDTA with other antimicrobials . The apparent synergy of mixtures of EDTA with quaternary ammonium surfactants (QAC) and specific antibiotics was successfully modelled . Minimum inhibitory concentrations (MIC) of the QAC and that of oxacillin and cefamandole were reduced by a factor of 3-10, whereas ampicillin was reduced by a factor of 70 from an MIC of 1524 to 21 mg l(-1) in the presence of 500 mg l(-1) of EDTA . CONCLUSIONS: A nonlinear concentration-dependent inhibition of P . aeruginosa by EDTA gives rise to apparent observation of synergy with other antimicrobials . SIGNIFICANCE AND IMPACT OF THE STUDY: This is a further example where the current methodology for the examination of antimicrobial synergy (the summed fractional inhibitory concentrations) leads to false conclusions.

Eye Contact Lens, 2004 Jan, 30(1), 42 - 3
Disinfection capacity of PuriLens contact lens cleaning unit against Acanthamoeba; Hwang TS et al.; PURPOSE: The PuriLens contact lens system is indicated for cleaning and disinfection of soft (hydrophilic) contact lenses by means of subsonic agitation to remove lens deposits and microorganisms, and ultraviolet irradiation of the storage solution for disinfection . The capacity of the PuriLens system to disinfect storage solutions contaminated with known concentrations of Staphylococcus aureus, Pseudomonas aeruginosa, and Acanthamoeba species was evaluated . METHODS: An in vitro assessment of the antibacterial and antiparasitic efficacy of the PuriLens system was performed . Separated batches of the storage solution for the cleansing system were contaminated with stock strains of S . aureus and P . aeruginosa . A comparison of the microbiologic content was made between the solution before and after the cycle . RESULTS: The PuriLens system effectively eradicated S . aureus and P . aeruginosa organisms after a 15-minute cycle . However, viable cysts of acanthamoeba were recovered in the solution after the 15-minute cycle . CONCLUSIONS: The PuriLens system is highly efficient in protecting against contamination with common bacterial ocular pathogens . Acanthamoeba cysts, however, can survive in the solution or contact lens bath undergoing integrated subsonic debridement and indirect ultraviolet light disinfection . Use of chemical disinfecting solutions that contain agents such as chlorhexidine or other cationic antiseptics may be advisable in conjunction with use of the PuriLens device, especially in high-risk settings.

Lancet Infect Dis, 2004 Jan, 4(1), 34 - 9
The changing face of malignant (necrotising) external otitis: clinical, radiological, and anatomic correlations; Rubin Grandis J et al.; Malignant (necrotising) external otitis is an invasive infection of the external auditory canal . Although elderly patients with diabetes remain the population most commonly affected, immunosuppressed individuals (eg, from HIV infection, chemotherapy, etc) are also susceptible to malignant external otitis . Pseudomonas aeruginosa is isolated from the aural drainage in more than 90% of cases . The pathophysiology is incompletely understood although aural water exposure (eg, irrigation for cerumen impaction) has been reported as a potential iatrogenic factor . The typical patient presents with exquisitely painful otorrhoea . If untreated, cranial neuropathies (most commonly of the facial nerve) can develop due to subtemporal extension of the infection . The diagnosis of malignant external otitis is based on a combination of clinical findings, an increased erythrocyte sedimentation rate, and radiographic evidence of soft tissue with or without bone erosion in the external canal and infratemporal fossa . Treatment consists of prolonged administration (6-8 weeks) of an antipseudomonal agent (typically an orally administered quinolone) . With the introduction and widespread use of both oral and topical quinolones, there are reports of less severe presentation of malignant external otitis and even the emergence of ciprofloxacin resistance . Reservation of systemic quinolones for the treatment of invasive ear infections is recommended.

Am J Respir Med, 2002, 1(2), 119 - 31
Diffuse panbronchiolitis: role of macrolides in therapy; Keicho N et al.; Diffuse panbronchiolitis (DPB) is characterized by chronic sinobronchial infection and diffuse bilateral micronodular pulmonary lesions consisting of inflammatory cells . Studies on disease etiology point to a genetic predisposition unique to Asians . Early therapy for DPB was largely symptomatic . The advent of macrolide antibiotics, including erythromycin, roxithromycin and clarithromycin, has strikingly changed disease prognosis . Low-dose, long-term macrolide therapy for DPB originated from detailed observations of response to therapy in a single patient . The bactericidal activity of macrolides, particularly erythromycin, is not a significant factor for their clinical efficacy in DPB . Firstly, irrespective of bacterial clearance, clinical improvement is observed in patients treated with erythromycin . Secondly, even in cases with bacterial superinfection with Pseudomonas aeruginosa resistant to macrolides, treatment has proved effective . Thirdly, the recommended dosage of macrolides produces peak levels in tissue that are below the minimum inhibitory concentrations for major pathogenic bacteria that colonize the airway . In the last two decades, the possible mechanism underlying the effectiveness of macrolide therapy has been extensively studied . The proposed mechanism of action includes inhibition of excessive mucus and water secretion from the airway epithelium, inhibition of neutrophil accumulation in the large airway, inhibition of lymphocyte and macrophage accumulation around the small airway, and modulation of bacterial virulence . The great success of macrolide therapy in diffuse panbronchiolitis may extend its application to the treatment of other chronic inflammatory disorders . If the anti-inflammatory activity of macrolides is independent of their bactericidal effect, new anti-inflammatory macrolides without antimicrobial activity should be developed to minimize emergence of macrolide-resistant micro-organisms.

Am J Respir Med, 2002, 1(4), 235 - 41
Macrolides in cystic fibrosis: is there a role?
Wolter JM, Seeney SL, McCormack JG.
A spectrum of anti-inflammatory properties, evidence of anti-infective action against Pseudomonas aeruginosa at sub-inhibitory concentrations and positive clinical experience in patients with diffuse panbronchiolitis, a disease with features in common with cystic fibrosis (CF), has prompted research to evaluate the role of macrolide therapy in patients with CF . Newer macrolides such as azithromycin have the advantage of improved tolerability and a prolonged intracellular half-life requiring an infrequent dosing regimen . Results from initial studies suggest a benefit from several months of macrolide therapy in patients with CF . An improvement in lung function was initially shown in a small open study in children, while maintenance of lung function compared with placebo, reduced acute respiratory exacerbations, and reduced systemic markers of inflammation were demonstrated in a randomized, placebo-controlled study of macrolide therapy in adult patients with CF . Additional controlled studies are required to determine optimal drug, dosage, and duration of therapy, and long-term adverse effects of prolonged therapy with macrolides in patients with CF . The potential, with long-term use, to induce resistance against other bacteria colonizing the upper respiratory tract e.g . pneumococci has not been explored . Measurement of cytokines and inflammatory mediators from the sputum of patients with CF is technically difficult and does not correlate with disease activity . There is a need for easily measurable, reproducible and clinically meaningful end-points for evaluation of new therapies in CF . The choice of appropriate outcome measures, apart from lung function, to monitor disease activity needs careful consideration in clinical trials determining the efficacy of macrolides in patients with CF . Evidence-based recommendations for the use of macrolides in the treatment of CF are not expected for some years although macrolides are already being prescribed for long-term use in some centers . There is a need for further research into mechanisms of anti-inflammatory action of macrolides in the lungs of patients with CF and whether or not such therapy may be beneficial in the long term.

J Assoc Physicians India, 2003 Oct, 51, 1021 - 2
Pacemaker endocarditis caused by Pseudomonas aeruginosa treated successfully; Chacko ST et al.; Infective endocarditis (IE) is a rare but serious complication of permanent cardiac pacemaker placement . Endocarditis in the presence of prosthetic valves and pacemakers is usually due to staphylococci . We present a case of pacemaker endocarditis caused by Pseudomonas aeruginosa that was successfully treated with a combination of antimicrobial therapy and percutaneous removal of the colonized lead.

Medicina (B Aires), 2003, 63(6), 715 - 20
{Effect of alcohol-gel hand hygiene on nosocomial infections due to multi-resistant Klebsiella pneumoniae}; Bermejo J et al.; Handwashing is considered the most important and effective infection control measure to prevent transmission of nosocomial pathogens . However, compliance with handwashing by health care workers is low . A new modality for hand hygiene is alcohol gel rub, which reduces time required, does not damage the skin and increases health care workers compliance . An observational study was conducted to assess the effect of alcohol-gel hand antiseptic on infection rates due to the 3 more frequent multi-resistant bacteria (Staphylococcus aureus, Klebsiella pneumoniae y Pseudomonas aeruginosa) in our hospital . Two periods were compared, 12 months before and 12 months after starting alcohol gel use . The second period (AG use) showed a significant reduction on incidence rates of Klebsiella pneumoniae with extended spectrum betalactamase (RR: 0.38) overall infections and specially bacteremias (RR: 0.10) . Nevertheless, on the basis of this study, we cannot conclude that the result was due to AG itself or to an increase in hand-hygiene compliance.

J Microencapsul, 2004 Feb, 21(1), 71 - 81
Design and in vitro evaluation of gentamicin-Eudragit microspheres intended for intra-ocular administration; Al-Kassas R; The conditions of preparation of gentamicin sulfate microspheres with high drug loading and a particle size less than 5 micro m, using a double-emulsion-solvent evaporation technique, intended for intra-ocular administration are described . The microspheres were prepared from poly methacrylate (Eudragit RS and RL) polymers cross-linked with polyvinyl alcohol . The parameters that improved the incorporation efficiency of gentamicin in the microspheres and controlled the particle size and surface morphology were investigated . Modifying the secondary aqueous phase by partially saturating it with various concentrations of either KCl or gentamicin increased the incorporation efficiency of the drug and affected the mean diameters of the microspheres . However, these characteristics were not altered when the initial drug loading was increased in the formulations . The modified gentamicin microspheres exhibited a smooth surface with an incorporation efficiency rate of 12.59% and a mean diameter of 4.06 micro m . The antimicrobial efficiency of gentamicin released from the modified particles against selected Gram-positive and -negative organisms including Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa confirmed that the entrapped gentamicin seemed to remain unaltered by the encapsulation process.

Clin Rev Allergy Immunol, 2003 Dec, 25(3), 233 - 47
Severe bronchiectasis; Morrissey BM et al.; Bronchiectasis is primarily the result of airway injury and remodeling attributable to recurrent or chronic inflammation and infection . The underlying etiologies include autoimmune diseases, severe infections, genetic abnormalities, and acquired disorders . Recurrent airway inflammation and infection may also be the result of allergic or immunodeficiency states such as allergic bronchopulmonary mycoses or HIV/AIDS . Bronchiectasis should be included in the differentiation diagnosis of any patient with chronic respiratory complaints such as cough and sputum production . Early clinical manifestations may be subtle . Hallmarks of severe bronchiectasis include fetid breath, chronic cough, and sputum production . The associated chronic respiratory infections and airway sepsis are punctuated by episodes of acute exacerbation . Prompt recognition and treatment of bronchiectasis may allow for prevention of disease progression and irreversible loss of lung function . This review of severe non-cystic fibrosis bronchiectasis describes the current pathophysiology, clinical presentations, and management of bronchiectasis . We review how impaired airway clearance and the inability to resolve infection and inflammation creates a vicious cycle of recurrent injury . The common clinical features of bronchiectasis and findings are presented and illustrated by radiographic images . The common species and significance of various organisms often recovered from the distal airways including: tuberculous and environmental mycobacteria, aspergillus, and bacteria such as Pseudomonas aeruginosa will be covered . Management strategies including sputum surveillance, sputum clearance, antimicrobial therapy including antifungal and antimyobacterial agents as well as the evidence for the use of inhalational and anti-inflammatory therapies such as corticosteroids are also discussed . Recommendations for the work-up and therapy of complications including hemoptysis and respiratory failure are presented.

Appl Microbiol Biotechnol, 2004 Jul, 65(1), 105 - 9 Epub 2004 Jan 09.
Bacillus amyloliquefaciens phage endolysin can enhance permeability of Pseudomonas aeruginosa outer membrane and induce cell lysis; Orito Y et al.; To determine the function of the C-terminal region of Bacillus amyloliquefaciens phage endolysin on Pseudomonas aeruginosa lysis, the permeabilization of the outer membrane of P . aeruginosa was analyzed . Glu-15 to His (E15H) and Thr-32 to Glu (T32E) substitutions were introduced into the Bacillus phage endolysin . Neither E15H nor T32E substitution induced enzymatic and antibacterial activities . These two, Glu-15 and Thr-32, were considered to be the active center of the enzyme . The addition of purified E15H and T32E proteins to P . aeruginosa cells induced the release of periplasmic beta-lactamase from the cells, indicating that both proteins enhance permeabilization of the outer membrane . However, the addition of E15H and T32E proteins to P . aeruginosa cells did not induce the release of cytoplasmic ATP from the cells . These results indicate that the antibacterial activity of the endolysin requires both the C-terminal enhancement of the permeabilization of the P . aeruginosa outer membrane and N-terminal enzymatic activity.

Biochemistry, 2004 Jan 13, 43(1), 183 - 94
Toward the elucidation of the catalytic mechanism of the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A; Armstrong S et al.; The catalytic mechanism for the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A was investigated by steady-state and stopped-flow kinetic analyses . The rate constants for binding of the NAD(+) substrate to the enzyme were found to be 4.7 +/- 0.4 microM(-1) s(-1) and 194 +/- 15 s(-1) for k(on) and k(off), respectively . The k(on) and k(off) rate constants for the eEF-2 substrate binding to the enzyme were 320 +/- 39 microM(-1) s(-1) and 131 +/- 22 s(-1), respectively . A potent, competitive inhibitor against the enzyme, 1,8-naphthalimide, bound the enzyme with k(on) and k(off) rates of 82 +/- 9 microM(-1) s(-1) and 51 +/- 6 s(-1), respectively . Furthermore, the binding on and off rates for the reaction products, ADP-ribose and nicotinamide, were too rapid for detection with the stopped-flow technique . Investigation of the pre-steady-state kinetics for the ADP-ribose transferase activity of the toxin-enzyme showed that there is no pre-steady-state complex formed during the catalytic cycle . Binding of NAD+ and smaller compounds representing the various parts of this substrate were investigated by the fluorescence quenching of the intrinsic toxin fluorescence . The binding data revealed a significant structural change in the enzyme upon NAD+ binding that could not be accounted for on the basis of the sum of the structural changes induced by the various NAD+ constituents . Product inhibition studies were conducted with nicotinamide and eEF-2-ADP-ribose, and the results indicate that the reaction involves a random-order ternary complex mechanism . Detailed kinetic analysis revealed that the eEF-2 substrate shows sigmoidal kinetic behavior with the enzyme, and fluorescence resonance energy transfer measurements indicated that wheat germ eEF-2 is oligomeric in solution.

Biochemistry, 2004 Jan 13, 43(1), 175 - 82
Single live cell imaging of chromosomes in chloramphenicol-induced filamentous Pseudomonas aeruginosa; Steel C et al.; Pseudomonas aeruginosa is a leading opportunistic pathogen in human infections, and it is renowned for its intrinsic resistance to structurally and functionally unrelated antibiotics . Filamentation induced by antibiotics appears to trigger bacteria to depart from a normal growth phase and enter a stationary growth phase . As antibiotic concentrations decline below a therapeutic range, filamentous bacteria begin to divide normally, leading to a more rapid regrowth of the bacteria . Furthermore, filamentous bacteria are associated with an increase in endotoxin release . Moreover, the immune system of a patient needs to cope with uncharacteristic filamentous bacteria . Thus, it is biologically and clinically significant to study and understand bacterial filamentation . In this study, we investigate the frequencies, conditions, and characteristics of a filamentous P . aeruginosa at single cell and single chromosome resolutions . Our results show that filamentous cells (elongated rods) contain multiple copies of the cell's chromosome . It appears that the unsuccessful segregation of replicated chromosomes in an individual cell accompanies the formation of undivided filamentous cells . The quantity of chromosomes and the length of the filamentous wild-type cells increase as the chloramphenicol concentration increases to 50 and 250 microg/mL, suggesting that chloramphenicol induces the filamentation . Filamentation in three strains of P . aeruginosa depends on the expression level of efflux pump (MexAB-OprM) and the minimum inhibitory concentration of chloramphenicol . This study also opens up the new possibility of real-time monitoring of modes of actions of antibiotics in live cells with both temporal and spatial resolution.

Biochemistry, 2004 Jan 13, 43(1), 140 - 7
Using nanoparticle optics assay for direct observation of the function of antimicrobial agents in single live bacterial cells; Kyriacou SV et al.; Multidrug resistance (MDR) has been reported in both prokaryotes and eukaryotes, underscoring the challenge of design and screening of more efficacious new drugs . For instance, the efflux pump of Pseudomonas aeruginosa (gram-negative bacteria) can extrude a variety of structurally and functionally diverse substrates, which leads to MDR . In this study, we present a new platform that studies modes of action of antibiotics in living bacterial cells (P . aeruginosa), in real-time, at nanometer scale and single-cell resolution using nanoparticle optics and single living cell imaging . The color index of silver (Ag) nanoparticles (violet, blue, green, and red) is used as the sized index (30 +/- 10, 50 +/- 10, 70 +/- 10, and 90 +/- 10 nm) for real-time measurement of sized transformation of the cell wall and membrane permeability at the nanometer scale . We have demonstrated that the number of Ag nanoparticles accumulated in cells increases as the aztreonam (AZT) concentration increases and as incubation time increases, showing that AZT induces the sized transformation of membrane permeability and the disruption of the cell wall . The results demonstrate that nanoparticle optics assay can be used as a new powerful tool for real-time characterization of modes of action of antimicrobial agents in living cells at the nanometer scale . Furthermore, studies of mutants of WT bacteria (nalB-1 and DeltaABM), suggest that an efflux pump (MexA-MexB-OprM) effectively extrudes substrates (nanoparticles) out of the cells, indicating that the MDR mechanism involves the induction of changes in membrane permeability and the intrinsic pump machinery.

Lung, 2003, 181(5), 237 - 44
Elevated tissue kallikrein activity in airway secretions from patients with tracheobronchitis associated with prolonged mechanical ventilation; O'Riordan TG et al.; The clinical course of patients undergoing prolonged mechanical ventilation is often complicated by the development of purulent tracheobronchitis . The purpose of this study was to assess whether ventilator-associated hypersecretion is associated with elevated levels of tissue kallikrein (TK) activity . TK can induce marked bronchial inflammation in animal models and TK activity is increased in the airway secretions of symptomatic asthmatics . It has not been studied in conditions with predominantly neutrophilic bronchial secretions, although animal data indicate that neutrophil elastase may stimulate TK activity . We measured TK activity in airway secretions of patients undergoing mechanical ventilation for more than 4 weeks (PMV group) and in two comparator groups: patients with cystic fibrosis, who were colonized with Pseudomonas aeruginosa (CF group) and patients undergoing mechanical ventilation for less than one week who did not have clinical evidence of purulent airway secretions (acute mechanical ventilation, AMV group) . We also compared the level of neutrophil elastase (NE) activity, an index of neutrophil activation, in the three patient groups . TK and NE activity in the sol phase were measured by the degradation of chromogenic substrates (DL Val-Leu-Arg pNA and N-Methoxy Succinyl Ala-Ala-Pro-Val pNA, respectively) . Intergroup differences in cell counts were not significant . However, TK activity was significantly less in the AMV group than in the PMV and cystic fibrosis patients (Kruskal-Wallis ANOVA, p < 0.05) . Elastase activity was significantly greater in the CF group (p < 0.05) than in the other two groups . Compared to patients undergoing short-term mechanical ventilation (AMV group), TK activity was elevated in patients with purulent tracheobronchitis associated with prolonged mechanical ventilation (PMV group) . The elevation in TK activity in these patients is comparable to levels in sputum from patients with cystic fibrosis (CF group), although the latter had a significantly higher level of NE activity . The observation of increased TK activity in patients with neutrophilic airway inflammation suggests that TK may play a role in modulating inflammation in ventilator-associated tracheobronchitis and may be worthy of further study to determine its source and significance.

J Chin Med Assoc, 2003 Oct, 66(10), 617 - 20
Pseudomonas aeruginosa endocarditis associated with endophthalmitis caused by arteriovenous fistula and graft infection; Hsu KH et al.; Bacterial endocarditis due to Pseudomonas aeruginosa arising from arteriovenous (AV) fistula and graft infection is unusual . We report an uncommon case of a 55-year-old woman housewife with chronic glomerulonephritis who had received hemodialysis (HD) for 5 years . She was admitted due to frequent episodes of AV fistula and graft infections in the past 5 years . She was admitted to our hospital because of a fever of unknown origin . During hospitalization, cardiac sonography and transesophageal echocardiography (TEE) confirmed a vegetation over the mitral valve . Blood culture yielded Pseudomonas aeruginosa . Endophthalmitis of the right eye was diagnosed by funduscopy because of painful redness of the right eye with exudative discharge . The patient was treated with ceftazidime for 9 weeks . Since then, she has been well, without any sequale after 1 year of following up . Physicians should be aware of the possibility of infective endocarditis in an uremic patient who suffers from fever of unknown origin . Early diagnosis with an adequate tool such as TEE and appropriate treatment will lead to an excellent prognosis.

Surg Endosc . 2003 Oct;17(10):1677.
Pyogenic cholangitis after inadvertent submucosal contrast injection in the papilla of Vater in a patient with cholestatic hepatitis; Katsinelos P et al.; Common bile duct stones and tumors constitute the leading cause of acute biliary tract obstruction and cholangitis . Septic complications after diagnostic endoscopic retrograde cholangiopancreatography (ERCP) are very unusual in unobstructed bile ducts . There are only three reported cases of patients without evidence of biliary tract disease who developed cholangitis and liver abscesses due to Pseudomonas aeruginosa . Biliary endoscopists believe that the inadvertent submucosal injection of contrast into the papilla of Vater is an innocent accident that has no serious consequences other than increasing the percentage of unsuccessful catheterizations of the common bile duct . Herein we describe a patient with drug-induced cholestatic hepatitis who developed pyogenic cholangitis after the inadvertent injection of submucosal contrast in the papilla of Vater.

J Bacteriol, 2004 Jan, 186(2), 518 - 34
Sequence analysis of the mobile genome island pKLC102 of Pseudomonas aeruginosa C; Klockgether J et al.; The Pseudomonas aeruginosa plasmid pKLC102 coexists as a plasmid and a genome island in clone C strains . Whereas the related plasmid pKLK106 reversibly recombines with P . aeruginosa clone K chromosomes at one of the two tRNA(Lys) genes, pKLC102 is incorporated into the tRNA(Lys) gene only close to the pilA locus . Targeting of the other tRNA(Lys) copy in the chromosome is blocked by a 23,395-bp mosaic of truncated PAO open reading frames, transposons, and pKLC102 homologs . Annotation and phylogenetic analysis of the large 103,532-bp pKLC102 sequence revealed that pKLC102 is a hybrid of plasmid and phage origin . The plasmid lineage conferred oriV and genes for replication, partitioning, and conjugation, including a pil cluster encoding type IV thin sex pili and an 8,524-bp chvB glucan synthetase gene that is known to be a major determinant for host tropism and virulence . The phage lineage conferred integrase, att, and a syntenic set of conserved hypothetical genes also observed in the tRNA(Gly)-associated genome islands of P . aeruginosa clone C chromosomes . In subgroup C isolates from patients with cystic fibrosis, pKLC102 was irreversibly fixed into the chromosome by the insertion of the large 23,061-bp class I transposon TNCP23, which is a composite of plasmid, integron, and IS6100 elements . Intramolecular transposition of a copy of IS6100 led to chromosomal inversions and disruption of plasmid synteny . The case of pKLC102 in P . aeruginosa clone C documents the intraclonal evolution of a genome island from a mobile ancestor via a reversibly integrated state to irreversible incorporation and dissipation in the chromosome.

J Biol Chem, 2004 Apr 2, 279(14), 14001 - 8 Epub 2003 Dec 30.
Structural insight into arginine degradation by arginine deiminase, an antibacterial and parasite drug target; Galkin A et al.; l-Arginine deiminase (ADI) catalyzes the irreversible hydrolysis of arginine to citrulline and ammonia . ADI is involved in the first step of the most widespread anaerobic route of arginine degradation . ADI, missing in high eukaryotes, is a potential antimicrobial and antiparasitic drug target . We have determined the crystal structure of ADI from Pseudomonas aeruginosa by the multi-wavelength anomalous diffraction method at 2.45 A resolution . The structure exhibits similarity to other arginine-modifying or substituted arginine-modifying enzymes such as dimethylarginine dimethylaminohydrolase (DDAH), arginine:glycine amidinotransferase, and arginine:inosamine-phosphate amidinotransferase, despite the lack of significant amino acid sequence homology to these enzymes . The similarity spans a core domain comprising five betabetaalphabeta motifs arranged in a circle around a 5-fold pseudosymmetry axis . ADI contains an additional alpha-helical domain of novel topology inserted between the first and the second betabetaalphabeta modules . A catalytic triad, Cys-His-Glu/Asp (arranged in a different manner from that of the thiol proteases), seen in the other arginine-modifying enzymes is also conserved in ADI, as well as many other residues involved in substrate binding . Based on this conservation pattern and the assumption that the substrate binding mode is similar to that of DDAH, an ADI catalytic mechanism is proposed . The main players are Cys-406, which mounts the nucleophilic attack on the carbon atom of the guanidinium group of arginine, and His-278, which serves as a general base.

Am J Ophthalmol, 2004 Jan, 137(1), 176 - 8
Orbital cellulitis, panophthalmitis, and ecthyma gangrenosum in an immunocompromised host with pseudomonas septicemia; Maccheron LJ et al.; PURPOSE: To describe a case of Pseudomonas aeruginosa septicemia complicated by orbital cellulitis, panophthalmitis, and ecthyma gangrenosum . DESIGN: Observational case report . METHODS: An immunosuppressed 62-year-old man developed an unusual skin rash and a painful, swollen right eye with decreased vision . He had myelodysplastic syndrome and P . aeruginosa septicemia . The skin rash manifested as ecthyma gangrenosum . Metastatic orbital cellulitis and panophthalmitis was diagnosed . RESULTS: Despite intravitreal and topical gentamicin, the patient eventually required enucleation . CONCLUSIONS: This case represents a rare combination of events: an immunocompromised man developed pneumonia, P . aeruginosa septicemia, and endogenous seeding of the Pseudomonas to the skin, orbit, and eye . Early recognition of endogenous ophthalmic disease is imperative . The prognosis of combined orbital cellulitis and panophthalmitis is poor.

J Electron Microsc (Tokyo), 2003, 52(5), 465 - 9
Effect of surface lipopolysaccharide on the nature of membrane vesicles liberated from the Gram-negative bacterium Pseudomonas aeruginosa; Nguyen TT et al.; Membrane vesicles (MVs) were isolated from three isogenic lipopolysaccharide (LPS) mutants derived from the Gram-negative bacterium Pseudomonas aeruginosa PAO1 and compared with the parent strain . Parent cells possessed serotype 05 (A+B+) LPS whereas the three mutants contained A+B-, A-B+ and A-B- . The MVs from the mutants contained their expected phenotypic LPS and varied in size, especially A-B- MVs . Mass and total protein differences were also noted . When the parent and mutant cells were treated with the outer membrane-perturbing antibiotic gentamicin, all cells produced 3-5 times more MVs and these had great variation in size, mass and total protein . It is concluded that the type of cellular LPS affects both the natural and gentamicin-induced development of MVs.

J Biol Chem, 2004 Mar 26, 279(13), 13166 - 73 Epub 2003 Dec 29.
The thrH gene product of Pseudomonas aeruginosa is a dual activity enzyme with a novel phosphoserine:homoserine phosphotransferase activity; Singh SK et al.; The thrH gene product of Pseudomonas aeruginosa has been shown to complement both homoserine kinase (thrB gene product) and phosphoserine phosphatase (serB gene product) activities in vivo . Sequence comparison has revealed that ThrH is related to phosphoserine phosphatases (PSP, EC 3.1.3.3) and belongs to the l-2-haloacid dehalogenase-like protein superfamily . We have solved the crystal structures of ThrH in the apoform and in complex with a bound product phosphate . The structure confirms an overall fold similar to that of PSP . Most of the catalytic residues of PSP are also conserved in ThrH, suggesting that similar catalytic mechanisms are used by both enzymes . Spectrophotometry-based in vitro assays show that ThrH is indeed a phosphoserine phosphatase with a K(m) of 0.207 mm and k(cat) of 13.4 min(-1), comparable with those of other PSPs . More interestingly, using high pressure liquid chromatography-based assays, we have demonstrated that ThrH is able to further transfer the phosphoryl group to homoserine using phosphoserine as the phosphoryl group donor, indicating that ThrH has a novel phosphoserine:homoserine phosphotransferase activity.

Bioorg Med Chem Lett, 2004 Jan 19, 14(2), 475 - 9
MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa . Part 3: Optimization of potency in the pyridopyrimidine series through the application of a pharmacophore model; Nakayama K et al.; The addition of substituents to the pyridopyrimidine scaffold of MexAB-OprM specific efflux pump inhibitors was explored . As predicted by a pharmacophore model, the incorporation substituents at the 2-position improved potency . Piperidines were found to be optimal, and further introduction of polar groups without compromising the activity was shown to be feasible . Careful positioning of the essential acidic moiety of the pharmacophore relative to the scaffold led to the discovery of vinyl tetrazoles with still greater potency.

Acta Paediatr, 2003 Nov, 92(11), 1267 - 71
Association between serum oncofetal antigens CA 19-9 and CA 125 and clinical status in patients with cystic fibrosis; Gronowitz E et al.; In cystic fibrosis (CF), mucus plugging in the airways and in the gastrointestinal tract leads to severe morbidity and mortality . The mucin-associated antigens CA 19-9 and CA 125 are markers of gastrointestinal malignancy, and CA 19-9 has also been reported in association with pulmonary function in CF . AIM: To test whether these antigens might serve as markers for the severity of pulmonary and gastrointestinal disease in CF . METHODS: In 99 patients, aged 1 to 48 y, serum levels of CA 19-9 and CA 125 were measured by RIA and ELISA and related to clinical data . RESULTS: Patients with severe mutations had significantly increased serum levels of CA 125, indicating an association with a more severe CF phenotype . This was further supported by the association with lung function, chronic pulmonary colonization of Pseudomonas aeruginosa and pancreatic insufficiency . CA 19-9 was also shown to be associated with lung function and Ps . aeruginosa colonization . No gastrointestinal malignancy was found in our patients despite very high values of CA 19-9 in some patients . During a 5-y follow-up, the very high serum levels of CA 19-9 decreased along with improved general condition of the patients . CONCLUSION: Increased serum levels of CA 125 in CF patients were associated with severe cystic fibrosis transmembrane conductance regulator mutations and a severe phenotype . Both antigens were associated with pseudomonas colonization and lung function and CA 125 also with pancreatic insufficiency . The estimates of CA 19-9 are hampered by the influence of the Lewis histo-blood group system on the synthesis of CA 19-9.

Glycobiology, 2004 Mar, 14(3), 1R - 15R Epub 2003 Dec 23.
Biosynthesis of 6-deoxyhexose glycans in bacteria; Maki M et al.; After the breakthroughs in genomic sequencing, one of the next challenges remains to understand the molecular biology of other classes of biomolecules, such as protein and lipids, many of which carry specific glycomodification when mediating their biological functions . This review focuses on the 6-deoxyhexose biosynthesis of cell surface glycans of three Gram-negative pathogens, Helicobacter pylori, Pseudomonas aeruginosa, and Actinobacillus actinomycetemcomitans serotype a . 6-Deoxysugars are important functional components of cell surface glycans, and their biosynthetic pathways might be suitable targets for novel interventions of antibacterial chemotherapy.

Br J Ophthalmol, 2004 Jan, 88(1), 25 - 8
The efficacy and safety of topical polymyxin B, neomycin and gramicidin for treatment of presumed bacterial corneal ulceration; Bosscha MI et al.; AIM: To evaluate the clinical efficacy and safety of topical polymyxin B, neomycin, and gramicidin for the treatment of suspected bacterial corneal ulceration at the Leiden University Medical Center . METHODS: Patients with a diagnosis of a suspected bacterial corneal ulcer between April 1995 and February 2002 were retrospectively identified and reviewed; clinical and microbiological features and response to therapy were analysed . All patients were treated with Polyspectran eye drops . RESULTS: In total, 91 patients were included in this analysis . Bacteriological cultures of 46 patients (51%) were positive and revealed 51 microorganisms . Staphylococcus aureus (29.4%) and Pseudomonas aeruginosa (23.5%) were the most frequently encountered bacteria . Eighteen patients switched therapy before complete healing of the corneal ulceration, four patients were lost to follow up . Of the 69 patients who completed Polyspectran treatment, re-epithelialisation occurred in 68 patients (99%) and on average took 12.6 (median 8) days . Among 91 patients, there were four perforations and one evisceration . Seven toxic or allergic reactions were reported . CONCLUSION: This study shows that the combination of polymyxin B, neomycin, and gramicidin is an effective and safe treatment of suspected corneal ulceration.

Antimicrob Agents Chemother, 2004 Jan, 48(1), 93 - 9
Immunomodulatory clarithromycin treatment of experimental sepsis and acute pyelonephritis caused by multidrug-resistant Pseudomonas aeruginosa; Giamarellos-Bourboulis EJ et al.; Clarithromycin was administered intravenously to 55 rabbits to evaluate its effect on experimental sepsis caused by multidrug-resistant Pseudomonas aeruginosa . Acute pyelonephritis was induced after ligation of the right ureter and injection of 10(8) CFU of the test isolate per kg of body weight into the renal pelvis . The animals were divided into six groups: group A, controls; group B, rabbits that received one intravenous dose of 80 mg of clarithromycin per kg concomitantly with bacterial challenge; group C, rabbits that received two doses of clarithromycin, the second one of which was given 2 h after the first one; group D, rabbits that received 15 mg of amikacin per kg; group E, rabbits that received one dose of clarithromycin and amikacin; and group F, rabbits that received two doses of clarithromycin and amikacin . Serum endotoxin levels were estimated by the QCL-1000 Limulus amoebocyte lysate assay, tumor necrosis factor alpha (TNF-alpha) levels were measured by a bioassay, and malondialdehyde (MDA) levels were measured by the thiobarbiturate assay . Viable bacterial counts in various tissue samples were also assessed . The mean survival times of the animals in groups A, B, C, D, E, and F were 4.50, 7.69, 4.07, 4.55, 11.55, and 11.60 days, respectively (P = 0.033 for group D versus group F, P = 0.006 for group D versus group E, P = not significant for group B versus group E, P = 0.042 for group C versus group F) . Serum endotoxin levels were similar between groups at all sampling times; TNF-alpha and MDA levels in groups B, C, E, and F decreased significantly over follow-up . The numbers of viable bacterial cells in the infected kidney were similar among the groups; those in the liver, spleen, lungs, and mesenteral lymph nodes were significantly decreased in groups B, E, and F compared to those in groups A and D . It is concluded that a prolongation of survival in animals with experimental sepsis caused by multidrug-resistant P . aeruginosa was achieved after coadministration of clarithromycin and amikacin and that the increased survival was probably attributable to the immunomodulatory properties of clarithromycin.

Burns, 2004 Feb, 30(1), 43 - 8
An evaluation of the role of systemic antibiotic prophylaxis in the control of burn wound infection at the Lagos University Teaching Hospital; Ugburo AO et al.; A prospective study was carried out on 61 patients to evaluate the role of systemic antibiotic prophylaxis in the control of burn wound infection . The patients were randomised into three groups: group 1 (n=21) received ampicillin and cloxacillin; group 2 (n=20) received erythromycin and genticin and a control group (n=20) received no systemic chemo prophylaxis . The burn wounds were similarly managed . Wound colonisation was determined from surface wound swab cultures and wound infection was determined from wound biopsy cultures and histopathology . The colonisation time (days) for the groups was 2.90+/-0.92, 3.15+/-0.77 and 3.05+/-0.83 for groups 1 and 2 and the control, respectively . The commonest organism isolated from contaminated wounds was Staphylococcus aureus . Wound infection was established in 5.70+/-1.70, 5.75+/-1.62 and 5.6+/-1.90 days for group 1, group 2 and the control group, respectively . There was no significant difference between wound infection time of control and group 1 nor was there such difference between the control and group 2 (P>0.05) . The commonest organism infecting burn wounds in all the groups was Pseudomonas aeruginosa followed by S . aureus . There was however a significant difference between the treatment groups and the control (P<0.05) with regard to the percentage of infected wounds that grew P . aeruginosa, compared to those that grew S . aureus . It was concluded that systemic antibiotic prophylaxis is of no value in controlling burn wound sepsis, and might even favour the growth of P . aeruginosa in the burn wounds.

Burns, 2004 Feb, 30(1), 3 - 26
Pseudomonas infections in the thermally injured patient; Tredget EE et al.; Pseudomonas aeruginosa, remains a serious cause of infection and septic mortality in burn patients, particularly when nosocomially acquired . A prototypic burn patient who developed serious nosocomially acquired Pseudomonas infection is described as an index case which initiated investigations and measures taken to identify the source of the infection . The effect of changes in wound care to avoid further nosocomial infections was measured to provide data on outcome and cost of care . The bacteriology of Pseudomonas is reviewed to increase the burn care providers understanding of the behaviour of this very common and serious pathogen in the burn care setting, before reviewing the approach to detection of the organism and treatment both medically and surgically . After controlling the nosocomial spread of Pseudomonas in our burn unit, we investigated the morbidity and mortality associated with nosocomial infection with an aminoglycoside resistant Pseudomonas and the associated costs compared to a group of case-matched control patients with similar severity of burn injury, that did not acquire resistant Pseudomonas during hospitalization at our institution . We found a significant increase in the mortality rate in the Pseudomonas group compared to controls . The morbidity in terms of length of stay, ventilator days, number of surgical procedures, and the amount of blood products used were all significantly higher in the Pseudomonas group compared to controls . Costs associated with antibiotic requirements were also significantly higher in the Pseudomonas group . Despite this increased resource consumption necessary to treat Pseudomonas infections, these efforts did not prevent significantly higher mortality rates when compared to control patients who avoided infection with the resistant organism . Thus, in addition to the specific measures required to identify and treat nosocomial Pseudomonas infections in burn patients, prevention of infection through modification of treatment protocols together with continuous infection control measures to afford early identification and eradication of nosocomial Pseudomonas infection are critical for cost-effective, successful burn care.

J Infect Chemother, 2003 Dec, 9(4), 297 - 303
Pretreatment of Pseudomonas aeruginosa with a sub-MIC of imipenem enhances bactericidal activity of neutrophils; Sasahara T et al.; The influence of the pretreatment of Pseudomonas aeruginosa strain O1 (PAO-1) with a sub-minimum inhibitory concentration (MIC) of imipenem on bactericidal activity, phagocytosis, the production of oxygen radical intermediates, and the induction of apoptosis in murine peritoneal neutrophils, as well as the catalase activity in the bacteria in comparison with that of ceftazidime-treated bacteria were studied . Bacteria treated with imipenem at (1/4) MIC were killed at significantly higher rates by neutrophils than ceftazidime-treated and nontreated bacteria . However, antibiotic-treated bacteria showed similar numbers of bacteria-phagocytized neutrophils to those in untreated bacteria . Imipenem pretreatment of bacteria led to an increase in the production of oxygen radical intermediates by neutrophils and the inhibition of neutrophilic apoptosis following incubation, whereas these features did not occur in neutrophils incubated with nontreated and ceftazidime-treated bacteria . The catalase activity of bacteria was not suppressed by pretreatment with either antibiotic at (1/4) MIC . These findings suggest that the exposure of P . aeruginosa to a sub-MIC of imipenem enhances the susceptibility of the bacteria to neutrophilic killing and effectively modifies the physiological activities of neutrophils, but does not decrease bacterial catalase activity . These actions may account for the postantibiotic leukocyte enhancement (PALE) effect of a sub-MIC of imipenem in the host.

Vet Rec, 2003 Dec 6, 153(23), 704 - 7
Purulent rhinitis and otitis caused by Pseudomonas aeruginosa in sheep showered with contaminated 'shower wash'; Watson PJ et al.; Three crossbred lowland ewes developed a severe purulent rhinitis and another three ewes developed a severe purulent otitis externa/media after being showered with a wash that had been used 24 to 48 hours before on a separate group of Cheviot ewes with lesions of dermatitis . Pseudomonas aeruginosa was isolated in pure growth from the aural and nasal abscesses and also from the dermatitis lesions . Extended antibiotic susceptibility testing and the random amplification of polymorphic DNA indicated that a single clonal type was associated with the rhinitis and otitis and with the dermatitis, providing strong evidence of an epidemiological link between the lesions of dermatitis and the aural and nasal abscesses through the use of the contaminated 'shower wash'.

Braz J Med Biol Res, 2004 Jan, 37(1), 77 - 82 Epub 2003 Dec 18.
Typing of Pseudomonas aeruginosa from hospitalized patients: a comparison of susceptibility and biochemical profiles with genotype; Freitas AL et al.; Typing techniques are essential for understanding hospital epidemiology, permitting the elucidation of the source of infection and routes of bacterial transmission . Although DNA-based techniques are the "gold standard" for the epidemiological study of Pseudomonas aeruginosa, antibiotic profiles and biochemical results are used because they are easy to perform and to interpret and relatively inexpensive . Antibiotypes (susceptibility profiles) and biotypes (biochemical profiles) were compared to genotypes established by DNA restriction enzyme analysis in 81 clinical isolates of P . aeruginosa from three hospitals in Porto Alegre, Brazil . The epidemiological relationship among patients was also evaluated . Susceptibility and restriction profiles were discrepant in more than 50% of the cases, and many antibiotypes were observed among isolates from the same genotype . Furthermore, susceptibility profiles did not allow the distinction of isolates from unrelated genotypes . Since a large number of isolates (63%) yielded the same biochemical results, only 10 biotypes were detected, showing that this typing method has a low discriminatory power . On the other hand, DNA restriction enzyme typing allowed us to establish 71 distinct types . Epidemiological data about the relation among P . aeruginosa isolates were not conclusive . The results of the present study indicate that the only method that can establish a clonal relation is DNA restriction enzyme typing, whereas the other methods may cause misleading interpretations and are inadequate to guide proper infection control measures.

J Pediatr (Rio J), 1994 Jul-Aug, 70(4), 243 - 6
{Kwashiorkor as early clinical manifestation in a baby with cystic fibrosis}; Damaceno N et al.; A case of cystic fibrosis in a baby presenting Kwashiorkor (edema, hypoalbuminemia and anemia ) is described . This is a very unusual presentation, easily attributed to unfavourable socio-economic conditions of the population, and classically considered a marker for severe pulmonary disease during the first year of life . Presence of severe pancreatic insufficiency is related to genotype delta F508 (homozygote) but colonization and early infection with Staphylococcus aureus and Pseudomonas aeruginosa in this baby requires further study . Cystic Fibrosis is the most frequent genetic disease among Caucasians, and early diagnosis has prognostic implications, agreed upon by all.

J Immunol, 2004 Jan 1, 172(1), 509 - 15
Neutrophil serine proteinases cleave bacterial flagellin, abrogating its host response-inducing activity; Lopez-Boado YS et al.; After bacterial infection, neutrophils dominate the cellular infiltrate . Their main function is assumed to be killing invading pathogens and resolving the inflammation they cause . Activated neutrophils are also known to release a variety of molecules, including the neutrophil serine proteinases, extracellularly . The release of these proteinases during inflammation creates a proteolytic environment where degradation of different molecules modulates the inflammatory response . Flagellin, the structural component of flagella on many bacterial species, is a virulence factor with a strong proinflammatory activity on epithelial cells and other cell types . In this study we show that both human and mouse neutrophil serine proteinases cleave flagellin from Pseudomonas aeruginosa and other bacterial species . More important, cleavage of P . aeruginosa flagellin by the neutrophil serine proteinases neutrophil elastase and cathepsin G resulted in loss of the biological activity of this virulence factor, as evidenced by the lack of innate host defense gene expression in human epithelial cells . The finding that flagellin is susceptible to cleavage by neutrophil serine proteinases suggests a novel role for these enzymes in the inflammatory response to infection . Not only can these enzymes kill bacteria, but they also degrade their virulence factors to halt the inflammatory response they trigger.

J Immunol, 2004 Jan 1, 172(1), 418 - 25
Localization of cystic fibrosis transmembrane conductance regulator to lipid rafts of epithelial cells is required for Pseudomonas aeruginosa-induced cellular activation; Kowalski MP et al.; The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein is an epithelial cell receptor for the outer core oligosaccharide of the Pseudomonas aeruginosa LPS . Bacterial binding leads to CFTR-dependent bacterial internalization, initiation of NF-kappaB nuclear translocation, cellular desquamation, and eventual apoptosis of the infected cells, all of which are critical for innate immune resistance to infection with this pathogen . Lack of this reaction in CF patients underlies their hypersusceptibility to chronic P . aeruginosa infection . In this study we tested whether these epithelial cell responses are dependent upon the localization of CFTR to lipid rafts . Confocal microscopy showed that green fluorescent protein-tagged CFTR (GFP-CFTR) and the lipid raft marker ganglioside GM1 colocalized at sites of P . aeruginosa contact and internalization . GFP-CFTR localized to low density Triton X-100-insoluble fractions in lysates of Madin-Darby canine kidney GFP-CFTR cells, and P . aeruginosa infection increased the levels of GFP-CFTR in these fractions as determined by Western blot . Cells expressing GFP-DeltaF508-CFTR did not have rafts with detectable CFTR protein . Extraction of cell surface cholesterol via cyclodextrin treatment of the cells inhibited CFTR entry into rafts . In addition, cyclodextrin treatment of both human and canine epithelial cells inhibited cellular ingestion of P . aeruginosa, NF-kappaB nuclear translocation, and apoptosis . These results indicate that lipid raft localization of CFTR is required for signaling in response to P . aeruginosa infection . Such signaling is needed for the coordination of innate immunity to P . aeruginosa lung infection, a process that is defective in CF.

J Immunol, 2004 Jan 1, 172(1), 398 - 409
Essential contribution of monocyte chemoattractant protein-1/C-C chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia; Amano H et al.; Neutrophil infiltration is the first step in eradication of bacterial infection, but neutrophils rapidly die after killing bacteria . Subsequent accumulation of macrophage lineage cells, such as alveolar macrophages (AMs), is essential to remove dying neutrophils, which are a source of injurious substances . Macrophage lineage cells can promote tissue repair, by producing potential growth factors including hepatocyte growth factor (HGF) . However, it remains elusive which factor activates macrophage in these processes . Intratracheal instillation of Pseudomonas aeruginosa caused neutrophil infiltration in the airspace; subsequently, the numbers of total AMs and neutrophil ingested AMs were increased . Bronchoalveolar lavage (BAL) fluid levels of monocyte chemoattractant protein (MCP)-1/CC chemokine ligand-2 (CCL2), a potent macrophage-activating factor, were increased before the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid . Immunoreactive MCP-1 proteins were detected in alveolar type II epithelial cells and AMs only after P . aeruginosa infection . The administration of anti-MCP-1/CCL2 Abs reduced the increases in the number of AM-ingesting neutrophils and HGF levels in BAL fluid, and eventually aggravated lung tissue injury . In contrast, the administration of MCP-1/CCL2 enhanced the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid, and eventually attenuated lung tissue injury . Furthermore, MCP-1/CCL2 enhanced the ingestion of apoptotic neutrophils and HGF production by a mouse macrophage cell line, RAW 267.4, in a dose-dependent manner . Collectively, MCP-1/CCL2 has a crucial role in the resolution and repair processes of acute bacterial pneumonia by enhancing the removal of dying neutrophils and HGF production by AMs.

Infect Immun, 2004 Jan, 72(1), 546 - 58
The ADP ribosyltransferase domain of Pseudomonas aeruginosa ExoT contributes to its biological activities; Garrity-Ryan L et al.; ExoT is a type III secreted effector protein found in almost all strains of Pseudomonas aeruginosa and is required for full virulence in an animal model of acute pneumonia . It is comprised of an N-terminal domain with GTPase activating protein (GAP) activity towards Rho family GTPases and a C-terminal ADP ribosyltransferase (ADPRT) domain with minimal activity towards a synthetic substrate in vitro . Consistent with its activity as a Rho family GTPase, ExoT has been shown to inhibit P . aeruginosa internalization into epithelial cells and macrophages, disrupt the actin cytoskeleton through a Rho-dependent pathway, and inhibit wound repair in a scrape model of injured epithelium . We have previously shown that mutation of the invariant arginine of the GAP domain to lysine (R149K) results in complete loss of GAP activity in vitro but only partially inhibits ExoT anti-internalization and cell rounding activity . We have constructed in-frame deletions and point mutations within the ADPRT domain in order to test whether this domain might account for the residual activity observed in ExoT GAP mutants . Deletion of a majority of the ADPRT domain (residues 234 to 438) or point mutations of the ADPRT catalytic site (residues 383 to 385) led to distinct changes in host cell morphology and substantially reduced the ability of ExoT to inhibit in vitro epithelial wound healing over a 24-h period . In contrast, only subtle effects on the efficiency of ExoT-induced bacterial internalization were observed in the ADPRT mutant forms . Expression of each domain individually in Saccharomyces cerevisiae was toxic, whereas expression of each of the catalytically inactive mutant domains was not . Collectively, these data demonstrate that the ADPRT domain of ExoT is active in vivo and contributes to the pathogenesis of P . aeruginosa infections.

Microb Pathog, 2004 Feb, 36(2), 59 - 66
Binding of plasminogen to Pseudomonas aeruginosa results in formation of surface-associated plasmin and enhanced bacterial invasiveness; da Silva CM et al.; The interaction of Pseudomonas aeruginosa with plasminogen (Plg) is herein reported . Plg bound similarly to laboratory and clinical P . aeruginosa isolates from blood of septicemic patients and stools of asymptomatic carriers . No difference in Plg capture was detected between the piliated PAK strain and its isogenic nonpiliated mutant . Western immunoblotting results suggested that low molecular weight nonpilus adhesins from the bacterial outer membranes accounted for the Plg capture . Bacteria-bound Plg was converted to bioactive plasmin in the presence of exogenous urokinase-type Plg activator . The presence of surface-bound plasmin enhanced significantly the P . aeruginosa capability to invade fibrin gels and a reconstituted basement membrane matrix . These findings support the concept that Plg capture by P . aeruginosa may represent a mechanism which offers advantages to bacterial invasiveness through tissue barriers.

Gesundheitswesen, 2003 Dec, 65(12), 736 - 7
{Pseudomonads in a new hospital building}; Behrends HB; Testing the piping of a new hospital showed that the drinking water was contaminated with Pseudomonas aeruginosa . Because of pollution the plumbing system was treated with oxidizing biocides and air-water flushing for several times, unfortunately without stopping the bacterial recontamination . The reasons were firstly stagnation due to dead legs in the piping system, oversized pipes, very low water exchange rates and the installation of a catch water system, secondly low flow velocity because of low water pressure . After these sources of recontamination had been repaired, chlorine dioxide sanitization could be stopped successfully

J Bacteriol, 2004 Jan, 186(1), 262 - 5
An H(+)-coupled multidrug efflux pump, PmpM, a member of the MATE family of transporters, from Pseudomonas aeruginosa; He GX et al.; We cloned the gene PA1361 (we designated the gene pmpM), which seemed to encode a multidrug efflux pump belonging to the MATE family, of Pseudomonas aeruginosa by the PCR method using the drug-hypersensitive Escherichia coli KAM32 strain as a host . Cells of E . coli possessing the pmpM gene showed elevated resistance to several antimicrobial agents . We observed energy-dependent efflux of ethidium from cells possessing the pmpM gene . We found that PmpM is an H(+)-drug antiporter, and this finding is the first reported case of an H(+)-coupled efflux pump in the MATE family . Disruption and reintroduction of the pmpM gene in P . aeruginosa revealed that PmpM is functional and that benzalkonium chloride, fluoroquinolones, ethidium bromide, acriflavine, and tetraphenylphosphonium chloride are substrates for PmpM in this microorganism.

J Bacteriol, 2004 Jan, 186(1), 248 - 52
Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide; Palma M et al.; Pseudomonas aeruginosa must often overcome a high concentration of oxidants to successfully infect the human host . We report here the results of a transcriptome profiling comparing cells treated with H(2)O(2) and untreated controls . The data indicate that the early response of P . aeruginosa to H(2)O(2) consists of an upregulation of protective mechanisms and a downregulation of primary metabolism.

Cell Microbiol, 2004 Jan, 6(1), 49 - 63
Cystic fibrosis airway epithelial cell polarity and bacterial flagellin determine host response to Pseudomonas aeruginosa; Hybiske K et al.; The role of epithelial polarity and bacterial factors in the control of the innate immune response of airway epithelial cells to Pseudomonas aeruginosa PAK was investigated using a human, nasal cystic fibrosis (DeltaF508/DeltaF508) epithelial cell line CF15 grown as confluent layers on permeable supports . Addition of PAK to the basal surface of CF15 layers caused significant expression changes in 1525 different genes (out of 12 625 examined), including the cytokines IL-6, IL-8, IL-1beta and TNF-alpha, as well as genes associated with leucocyte adhesion, antibacterial factors, and NF-kappaB signalling . Confocal microscopy showed that nuclear migration of NF-kappaB in all CF15 cells was preceded by PAK binding to the basal and lateral surfaces of some cells . Addition of PAK to the apical surface of CF15 monolayers elicited changes in expression of only 602 genes, including 256 not affected during basolateral PAK exposure . Over time, cytokine expression during apical PAK was similar to that exhibited by basal PAK, but the magnitudes during apical treatment were much smaller with little/no nuclear migration of NF-kappaB in CF15 cells . Furthermore, these responses depended on the presence of flagellin, but not pili on the bacteria . Thus, P . aeruginosa triggered a strong innate immune response that depended on the apical versus basolateral polarity of CF15 cells and the presence of flagellin on the bacteria.

Tex Heart Inst J, 2003, 30(4), 322 - 4
Mycotic ascending aortic pseudoaneurysm secondary to pseudomonas mediastinitis at the aortic cannulation site; Vrochides D et al.; During the last 5 years, postoperative Pseudomonas mediastinitis has occurred in 2 of the 3,072 patients in our institution who have undergone cardiopulmonary bypass cardiac operations via a sternotomy . To our knowledge, there is no prior report in the English-language literature of postoperative Pseudomonas mediastinitis that originated at the aortic cannulation site, yet that was the site of origin in both of these patients . The 1st patient developed a mycotic pseudoaneurysm of the ascending aorta at the cannulation site, secondary to the development of Pseudomonas mediastinitis following aortic valve replacement . This sequela was successfully treated by means of aneurysmectomy and closure of the aorta with a bovine pericardial patch, under cardiopulmonary bypass with circulatory arrest . The 2nd patient developed pseudoaneurysm and perforation of the aorta at the cardioplegia needle site, secondary to Pseudomonas mediastinitis following aortic and mitral valve replacement . This patient died . In both patients, the cannulation site and the cardioplegia needle site had been closed with pledgeted sutures . Pseudomonas aeruginosa was cultured from both sites . Once the diagnosis of Pseudomonas mediastinitis is made following heart surgery, the patient should undergo reoperation, if possible, for removal of the foreign bodies (pledgeted sutures) . In addition, these patients should be monitored with chest magnetic resonance angiography every 3 months for 1 year, in order to diagnose early development of a mycotic pseudoaneurysm and subsequent complications.

Biochemistry, 2003 Dec 23, 42(50), 14752 - 61
Crystal structure at 1.45 A resolution of alanine racemase from a pathogenic bacterium, Pseudomonas aeruginosa, contains both internal and external aldimine forms; LeMagueres P et al.; The structure of the catabolic alanine racemase, DadX, from the pathogenic bacterium Pseudomonas aeruginosa, reported here at 1.45 A resolution, is a dimer in which each monomer is comprised of two domains, an eight-stranded alpha/beta barrel containing the PLP cofactor and a second domain primarily composed of beta-strands . The geometry of each domain is very similar to that of Bacillus stearothermophilus alanine racemase, but the rotation between domains differs by about 15 degrees . This change does not alter the structure of the active site in which almost all residues superimpose well with a low rms difference of 0.86 A . Unexpectedly, the active site of DadX contains a guest substrate that is located where acetate and propionate have been observed in the Bacillus structures . It is modeled as d-lysine and oriented such that its terminal NZ atom makes a covalent bond with C4' of PLP . Since the internal aldimine bond between the protein lysine, Lys33, and C4' of PLP is also unambiguously observed, there appears to be an equilibrium between both internally and externally reacted forms . The PLP cofactor adopts two partially occupied conformational states that resemble previously reported internal and external aldimine complexes.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003, 38(12), 2895 - 908
Disinfection of hospital wastewater by continuous ozonization; Chiang CF et al.; The disinfection of hospital wastewaters using the ozonization process was studied . The concentrations of ozone required to reach a sudden drop of coliform and Pseudomonas aeruginosa in the wastewater are 4.0-7.0 and 3.0-5.0 mg L(-1), respectively . For the hospital wastewater, the disinfection efficiencies were 0.518S(-1.1) for coliforms, 0.509S(-1.06) for Pseudomonas aeruginosa and 0.254S-(1.54) for total count, respectively . As to the effects of ozone input methods on the disinfection efficiency, the continuous ozonization process was ten times higher than the batch input process . The low COD removal rate was obtained at 25.0 mgL(-1) of ozone concentration for hospital wastewater . However, more biodegradable compounds resulted in the treated mixture.

Arch Pediatr, 2003 Sep, 10 Suppl 2, 376s - 379s
{Future prospects in the management of cystic fibrosis}; Pin I et al.; Basic and clinical research in cystic fibrosis have led to several new hypothesis to improve the management of the disease . The numerous tracks for new therapies may be explained by the lack of firm patho-physiological explanations for the disease and of knowledge of the best targets to get a significant improvement of the patients . After initial great hopes, there has been important limitations and slow down of gene therapy, imposing to go back to research programs on new vectors . New hopes have arisen with protein therapies, including chaperones molecules that can activate mutated CFTR proteins within the cells . New anti-inflammatory therapies are developed, including proteases inhibitors . The prevention of airway colonisation with Pseudomonas aeruginosa is fundamental and could go through the development of specific vaccines, cellular therapies or molecules directly acting on the virulent factors of the bacteria.

Arch Pediatr, 2003 Sep, 10 Suppl 2, 352s - 357s
{Antibiotic therapy against Pseudomonas aeruginosa and other gram-negative bacteria in cystic fibrosis}; Le Bourgeois M; In cystic fibrosis patients, bronchial infection with Pseudomonas aeruginosa (PA) is frequent, occurring often early in life . It becomes chronic because of a particular relationship between bacteria and host . Intensive antibiotic treatment often permits a transient eradication . When infection becomes chronic, antibiotics must be prescribed in a way to afford resistance . Efficacy of nebulized antibiotics is now recognized . Problems persist . Because of the difficulty to eradicate PA, new strategies may consist in diminishing the pathogenicity of PA, by inhibiting adhesion and virulence factors' production.

Arch Pediatr, 2003 Sep, 10 Suppl 2, 347s - 351s
{Antibacterial therapy outside of Pseudomonas aeruginosa}; Sardet A; Hemophilus influenzae and Staphylococcus aureus (SA) are the predominant pathogens in infants with cystic fibrosis (CF) . SA was the major cause of death in the pre-antibiotic era . The reason of the association of SA in CF is unclear . SA causes early damage of the respiratory tract and paves the way for Pseudomonas aeruginosa (PA) . Based of this hypothesis, some centers use prophylactic antibiotics, but their efficacy is not proved and may favor growth of PA . Treatment of exacerbations is mandatory . Oral antibiotics are preferred in most cases, although few controlled clinical studies have been reported . Emergence of methicillin-resistant Staphylococcus aureus (MRSA) strains appeared during the recent years . Treatment of MRSA is difficult, patients segregation is discussed.

Acta Otorrinolaringol Esp, 2003 Aug-Sep, 54(7), 485 - 90
{Use of topical ciprofloxacin in chronic suppurating otitis media}; Ramos A et al.; OBJECTIVE: A prospective study is presented . We carry out an analysis of the results obtained after treatment with different protocols of administration of ciprofloxacin, during the active phase in chronic otitis media and in chronic otorrhea . MATERIAL AND METHOD: A multicenter, prospective study is carried out, in 3 ENT departments, corresponding to 3 reference tertiary hospitals . 300 patients were included ranging from 5 to 73 years old, all were diagnosed of chronic disease of the middle ear: simple chronic otitis media (n = 128), chronic otitis with bone reabsorption (n = 57), cholesteatoma infection (n = 42) and postsurgery ear infection (n = 73) . Patients were placed in 5 treatment groups: ciprofloxacin (oral administration) (only adults were included), topical ciprofloxacin (0.5%), topical ciprofloxacin (0.2%), topical ciprofloxacin (0.2%) plus oral ciprofloxacin and ciprofloxacin (0.3%) plus topical fluocinolone . There was a control group treated with polimixin plus neomicine and hidrocortison . RESULTS: The most common isolated bacterias were: Pseudomonas aeruginosa and S . aureus . We found 19 resistant strains to ciprofloxacin . A better therapeutic response was observed in the topical administration groups . In topical administration, a difference was only observed in the cholesteatoma and chronic middle ear infection with bone reabsorption groups, in those patients that were administered the ciprofloxacin with fluocinolone . CONCLUSION: Forms of topical treatment, with ciprofloxacin, in active infection of chronic disease of the middle ear, improve the results compared to oral administration . The association with of topical fluocinolone improves the results in the cases with cholesteatoma infection and chronic middle ear infection.

Chemotherapy, 2003 Dec, 49(6), 294 - 7
In vitro activity of combination therapy with cefepime, piperacillin-tazobactam, or meropenem with ciprofloxacin against multidrug-resistant Pseudomonas aeruginosa strains; Erdem I et al.; The prevalence of multidrug-resistant Pseudomonas aeruginosa strains has been increasing every year, and treatment with various antimicrobial combinations has been offered alternatively in the clinical practice . The aim of this study was to evaluate the in vitro effects of combinations of meropenem, cefepime, or piperacillin-tazobactam with ciprofloxacin against multidrug-resistant P . aeruginosa strains by the time-kill method . The results show that both the combination of two beta lactams with ciprofloxacin and three beta lactams with ciprofloxacin against 4 of 5 multidrug-resistant P . aeruginosa strains has no effect in vitro . The combination of one beta lactam (meropenem, cefepime, piperacillin-tazobactam) with ciprofloxacin has a synergistic effect against one strain of multidrug-resistant P . aeruginosa that is ciprofloxacin susceptible and resistant to meropenem, cefepime, and piperacillin-tazobactam . None of the combinations had an antagonistic effect against these multidrug-resistant strains .

Am J Respir Crit Care Med, 2004 Apr 1, 169(7), 811 - 5 Epub 2003 Dec 11.
Adult cystic fibrosis exacerbations and new strains of Pseudomonas aeruginosa; Aaron SD et al.; We hypothesized that in adults with cystic fibrosis, the acquisition of a new strain of Pseudomonas aeruginosa may be associated with a pulmonary exacerbation . Eighty-four patients who were chronically infected with P . aeruginosa were prospectively followed from eight centers over a 26-month period . Patients had sputum cultures performed every 3 months while clinically stable and at the time of an exacerbation . Forty patients (48%) had an exacerbation requiring intravenous antibiotics during the study period, and in 36 of these patients, their P . aeruginosa isolates were genetically typeable by pulsed-field gel electrophoresis . In 34 of the 36 patients (94%), P . aeruginosa recovered during clinical stability and at exacerbation were of the same genotype . In only two patients (6%; 95% confidence interval, 0-18%) was a new P . aeruginosa clone cultured during an exacerbation that had not been cultured during clinical stability . There were no significant differences in antibiotic susceptibilities, measured as mean minimal inhibitory concentrations, for isolates retrieved during clinically stable periods compared with isolates retrieved during exacerbations . We conclude that for the majority of adult patients with cystic fibrosis a new pulmonary exacerbation is not caused by the acquisition of a new strain of P . aeruginosa.

Am J Respir Crit Care Med, 2004 Mar 15, 169(6), 679 - 86 Epub 2003 Dec 11.
Preferential diaphragmatic weakness during sustained Pseudomonas aeruginosa lung infection; Divangahi M et al.; Infection with Pseudomonas aeruginosa plays a major role in the pulmonary inflammation and injury associated with cystic fibrosis . Lung inflammation may also lead to more widespread systemic effects on other organs . We tested the following hypotheses: (1) ongoing P . aeruginosa lung infection produces diaphragmatic and limb muscle weakness and (2) such muscle dysfunction is directly correlated with the level of pulmonary inflammation . Chronic bronchopulmonary infection with mucoid P . aeruginosa was induced in C57BL/6 mice . At Day 2 after infection, diaphragmatic force was decreased (37%) only in mice infected with a high dose of 1 x 10(6) cfu, whereas by Day 7 after infection, diaphragmatic force was similarly reduced (36%) even at a fivefold lower inoculating dose . No significant correlations were found between diaphragmatic weakness and pulmonary inflammation, as assessed by the number of neutrophils, macrophages, and lymphocytes in bronchoalveolar lavage fluid . Moreover, in marked contrast to the diaphragm, no effects of P . aeruginosa infection on contractile function were observed in prototypical slow- and fast-twitch hindlimb muscles . We conclude that sustained lung infection with P . aeruginosa induces preferential weakness of the diaphragm, which is not directly correlated with the degree of pulmonary inflammation induced under these conditions.

Am J Respir Crit Care Med, 2004 Mar 1, 169(5), 645 - 53 Epub 2003 Dec 11.
Cytokine secretion by cystic fibrosis airway epithelial cells; Becker MN et al.; It is controversial whether mutations in cystic fibrosis transmembrane conductance regulator intrinsically dysregulate inflammation . We characterized passage 2 human tracheobronchial epithelial cell cultures morphologically and physiologically and determined whether cytokine production or nuclear factor-kappaB activation was systematically altered in cystic fibrosis (CF) cells . Non-CF and CF cells originating from a total of 33 and 25 lungs, respectively, were available for culture on plastic or at an air-liquid interface until well differentiated . Forskolin-stimulated short-circuit currents were present in representative polarized non-CF cultures and were absent in CF cultures, whereas uridine 5'-triphosphate-stimulated currents were present in both . Constitutive or interleukin (IL)-1beta-induced IL-8 or IL-6 secretion or nuclear factor-kappaB activity was not significantly different between non-CF and CF cells . The cytokines regulated upon activation, normal T cell expressed and secreted (RANTES) and IL-10 were not detectable . Stimulation with tumor necrosis factor-alpha or a synthetic toll-like receptor 2 agonist or variable doses and times of Staphylococcus aureus culture filtrate revealed a single dose- and time-dependent difference in IL-8 production by CF cells . Interestingly, although IL-8 secretion after stimulation with Pseudomonas aeruginosa filtrates was not greater in CF cells in the absence of human serum, it was variably greater in its presence . Thus, although exaggerated responses may develop under certain conditions, our results do not support an overall intrinsically hyperinflammatory phenotype in CF cells.

Carbohydr Res, 2003 Nov 14, 338(23), 2667 - 77
Structure of a highly phosphorylated lipopolysaccharide core in the Delta algC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3); Kooistra O et al.; Lipopolysaccharides (LPS) were isolated from rough-type mutant strains of Pseudomonas aeruginosa (Delta algC) derived from wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3) . Structural studies of the LPS core region with a special focus on the phosphorylation pattern were performed by 2D NMR spectroscopy, including a 1H,(31)P HMQC-TOCSY experiment, MALDI-TOF MS, and Fourier-transform ion cyclotron resonance ESIMS using the capillary skimmer dissociation technique . Both LPS were found to contain two residues each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep), one residue of N-(L-alanyl)-D-galactosamine and one O-carbamoyl group (Cm) on the distal Hep residue . The following structures of a tetrasaccharide trisphosphate from strain PAC1R Delta algC and that carrying an additional ethanolamine phosphate group (PEtN) from strain PAO1 Delta algC were elucidated: {carbohydrate structre: see text} where R=P in PAC1R Delta algC and PPEtN in PAO1 Delta algC . To our knowledge, in this work the presence of ethanolamine diphosphate is unambiguously confirmed and its position established for the first time in the LPS core of a rough-type strain of P . aeruginosa . In addition, the structure of the complete LPS core of wild-type strain P . aeruginosa PAO1 was reinvestigated and the position of the phosphorylation sites was revised.

Carbohydr Res, 2003 Nov 14, 338(23), 2549 - 56
Promises and pitfalls of Pseudomonas aeruginosa lipopolysaccharide as a vaccine antigen; Pier GB; Antibodies directed to the Pseudomonas aeruginosa lipopolysaccharide (LPS) O-antigens have clearly shown to mediate the most effective immunity to infection caused by LPS-smooth strains . Such strains are major causes of disease in immunocompromised hosts such as burn or cancer patients, individuals in intensive care units, and those who utilize extended-wear contact lenses . Yet producing an effective vaccine composed of non-toxic, immunogenic polysaccharides has been challenging . The chemical diversity among the different O-antigens representative of the 20 major serotypes, plus additional diversity among some O-antigens representing variant subtype antigens, translates into a large degree of serologic variability that increases the complexity of O-antigen specific vaccines . Further complications come from the poor immunogenicity of the major protective epitope expressed by some O-antigens, and a large degree of diversity in animal responses that preclude predicting the optimal vaccine formulation from such studies . Nonetheless human trials over the years of vaccines eliciting O-antigen immunity have been encouraging, though no vaccine has yet been fully evaluated and found to be clinically efficacious . Newer vaccine approaches such as using polysaccharide-protein conjugates and passive therapy with monoclonal or polyclonal immune sera offer some additional means to try and produce an effective immunotherapeutic reagent for this problematic pathogen.

Spectrochim Acta A Mol Biomol Spectrosc, 2004 Jan, 60(1-2), 271 - 7
Derivatives of phosphate Schiff base transition metal complexes: synthesis, studies and biological activity; Abd El-Wahab ZH et al.; We report the synthesis and structural characterization of series of tetra- and hexacoordinate metal chelate complexes of phosphate Schiff base ligands having the general composition LMX(n).H(2)O and L(2)MX(n) (L=phosphate Schiff base ligand; M=Ag(+), Mn(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+), or Fe(3+) and X=NO(3)(-), Br(-) or Cl(-)) . The structure of the prepared compounds was investigated using elemental analysis, IR, 1H and 31P NMR, UV-vis, mass spectra, solid reflectance, magnetic susceptibility and conductance measurements as well as conductometric titration . In all the complexes studied, the ligands act as a chelate ligand with coordination involving the phosphate-O-atom and the azomethine-N-atom . IR, solid reflectance spectra and magnetic moment measurement are used to infer the structure and to illustrate the coordination capacity of ligand . IR spectra show the presence of coordinated nitrate and water molecule, the magnetic moments of all complexes show normal magnetic behavior and the electronic spectra of the metal complexes indicate a tetra- and octahedral structure for Mn(2+), octahedral structure of Fe(3+) and both square-planar and distorted octahedral structure for Cu(2+) complexes . Antimicrobial activity of the ligands and their complexes were tested using the disc diffusion method and the chosen strains include Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Microsporum canis, Trichophyton mentagrophyte and Trichophyton rubrum . Some known antibiotics are included for the sake of comparison and the chosen antibiotic are Amikacin, Doxycllin, Augmantin, Sulperazon, Unasyn, Septrin, Cefobid, Ampicillin, Nitrofurantion, Traivid and Erythromycin.

Lancet, 2003 Dec 6, 362(9399), 1888 - 93
Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa; Yokoyama K et al.; BACKGROUND: Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase . These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity . Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically . We aimed to clone and characterise the genetic determinant of this resistance . METHODS: We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen . PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997 . FINDINGS: An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2 . The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin . The 756-bp nucleotide rmtA gene encoded a protein, RmtA . This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation . Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA . Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses . INTERPRETATION: Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa . Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.

Math Med Biol, 2003 Sep, 20(3), 227 - 60
Modelling host tissue degradation by extracellular bacterial pathogens; King JR et al.; Extracellular bacterial pathogens such as Pseudomonas aeruginosa are able to penetrate into host tissues (given an initial breech in the outer barrier, e.g . a wound) through the action of exo-toxins and degradative exo-enzymes . A mathematical model of this process is presented which, in the absence of significant immune response, predicts the progression of the bacteria into the tissue as a travelling wave whose velocity can be determined explicitly in terms of the model parameters . Simple in vitro experiments in protein-based matrices are performed which yield results consistent with this behaviour . A complementary in vitro experimental system with distinct qualitative behaviour is also studied, giving further insight and confidence in the modelling approach.

Chest, 2003 Dec, 124(6), 2239 - 43
Incidence, etiology, and outcome of nosocomial pneumonia in ICU patients requiring percutaneous tracheotomy for mechanical ventilation; Rello J et al.; OBJECTIVE: To determine the epidemiology of pneumonia in patients with tracheotomy receiving short-term mechanical ventilation . DESIGN: Observational prospective study . SETTING: A 14-bed medical-surgical ICU . SUBJECTS: Ninety-nine critically ill acute patients requiring percutaneous dilatational tracheotomy for mechanical ventilation . INTERVENTIONS: Tracheal aspirate obtained 48 h before tracheotomy . MEASUREMENTS AND MAIN RESULTS: Eighteen patients (18.1%) acquired pneumonia (median of 7 days after tracheotomy) . Pseudomonas aeruginosa was the most frequently identified pathogen, found in eight of the episodes (four not documented by prior tracheal colonization), followed by other Gram-negative bacilli . The development of ventilator-associated pneumonia (VAP) was not anticipated by any clinical variable . A positive tracheal aspirate (TA) culture result obtained before tracheotomy was associated with a risk of acquiring pneumonia of 19.7%, whereas sterile TA cultures were associated with a risk of 14.3% (p > 0.20) . VAP prolonged ICU stay or the ventilation period for a median of 19 days and 15 days, respectively . Overall mortality was 34.3%, but the presence of VAP did not increase the mortality rate . CONCLUSIONS: Percutaneous tracheotomy in patients receiving short-term mechanical ventilation predisposes to pneumonia . Pneumonia was associated with prolonged ventilation and ICU stay, but was not associated with increased mortality . Pseudomonas is a common pathogen after tracheotomy, and this observation should be considered in selecting an antibiotic regimen, because TA obtained prior to the tracheotomy often failed to identify this pathogen.

J Med Microbiol, 2004 Jan, 53(Pt 1), 73 - 81
Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patients compared with those of isolates from other origins; Lanotte P et al.; In order to improve our understanding of the colonization of the pulmonary tract of cystic fibrosis (CF) patients by Pseudomonas aeruginosa, 162 isolates from five different ecological origins were studied . The genetic features of each isolate were determined by random amplification of polymorphic DNA (RAPD) and by searching for eight virulence genes (six known virulence genes, algD, lasB, toxA, plcH, plcN and exoS, and two genes encoding putative neuraminidases, nan1 and nan2) . Five RAPD groups were identified . Most of the CF isolates were distributed equally in three of these groups (RA, RB and RC) . The CF isolates in RB were related to isolates from a wide variety of origins . The CF isolates in RA were related to a population composed of 65 % of the non-CF isolates from pulmonary tract infections . RC was mainly composed of CF isolates that were related to 30 % of isolates from plants . All genes except exoS and nan1 were present in all isolates . The exoS and nan1 virulence factor genes were most prevalent in CF isolates . exoS, which encodes exoenzyme S, was present in 94 % of CF isolates but also in 80 % of non-CF isolates from pulmonary tract infections . nan1, which encodes a putative neuraminidase, was found in 82.5 % of the isolates from group RC, which was composed largely of CF isolates . In conclusion, three major genogroups of P . aeruginosa isolates, each of which exhibits peculiar genetic features, are able to colonize CF patients . This may have different consequences on the outcome of pulmonary disease.

J Clin Microbiol . 2003 Dec;41(12):5741.
Pseudomonas cross-infection from cystic fibrosis patients to non-cystic fibrosis patients: implications for inpatient care of respiratory patients; Robinson P et al.; A 14-year-old boy with bronchiectasis secondary to chronic aspiration developed multiresistant Pseudomonas aeruginosa lower respiratory disease following several inpatient periods where accommodation and physiotherapy services were shared with cystic fibrosis (CF) patients known to be infected with the genetically identical strain of P . aeruginosa . Cross-infection with P . aeruginosa between CF patients and non-CF patients has not previously been described, and this finding raises significant issues relevant to the treatment of patients with non-CF suppurative lung disease.

Chembiochem, 2003 Dec 5, 4(12), 1317 - 25
Adhesion inhibition of F1C-fimbriated Escherichia coli and Pseudomonas aeruginosa PAK and PAO by multivalent carbohydrate ligands; Autar R et al.; In order to evaluate their inhibition of bacterial adhesion, the carbohydrate sequences GalNAcbeta1-->4Gal and GalNAcbeta1-->4Galbeta1-->4Glc were synthesized . The disaccharide was conjugated to dendrons based on the 3,5-di-(2-aminoethoxy)-benzoic acid branching unit to yield di- and tetravalent versions of these compounds . A divalent compound was also prepared that had significantly longer spacer arms . Relevant monovalent compounds were prepared for comparison . Their anti-adhesion properties against F1C-fimbriated uropathogenic Escherichia coli were evaluated in an ELISA-type assay by using a recombinant strain and also by using Pseudomonas aeruginosa strains PAO and PAK . Adhesion inhibition was observed in all cases, and multivalency effects of up to one order of magnitude were observed . The combination of spacer and multivalency effects led to a 38-fold increase in the potency of a divalent inhibitor with long spacer arms towards the PAO strain when compared with the free carbohydrate.

J Biosci, 2003 Dec, 28(6), 723 - 31
Reconfirmation of antimicrobial activity in the coelomic fluid of the earthworm Eisenia fetida andrei by colorimetric assay; Pan W et al.; A novel tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) was used in the assessment of antimicrobial activity in earthworm in the presence of phenazine methosulphate (PMS) as an electron coupling reagent . This activity was purified from the coelomic fluid of the earthworm (ECF), Eisenia fetida andrei (Oligochaeta, Lumbricidae, annelids) using a series of column chromatography techniques and was tested against three Gram-negative strains of Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and three Gram-positive strains of Staphylococcus aureus, Bacillus megaterium, Arthrobacter sp., respectively . Only the pigment-free eluate of coelomic fluid of the earthworm (ECFPE) showed activity against B . megaterium amongst three isolated active fractions . The anion (DEAE-52) exchange effluent of the ECFPE was reported to have the strongest activity against P . aeruginosa amongst the three active fractions . The 20% acetonitrile eluate (AE) by Sep-Pak C18 cartridge was also tested and showed fair resistance against E . coli, P . aeruginosa and Arthrobacter sp., respectively.

Appl Environ Microbiol, 2003 Dec, 69(12), 7273 - 80
Characterization of the argA gene required for arginine biosynthesis and syringomycin production by Pseudomonas syringae pv . syringae; Lu SE et al.; Two types of necrosis-inducing lipodepsipeptide toxins, called syringomycin and syringopeptin, are major virulence factors of Pseudomonas syringae pv . syringae strain B301D . A previous study showed that a locus, called syrA, was required for both syringomycin production and plant pathogenicity, and the syrA locus was speculated to encode a regulator of toxin production . In this study, sequence analysis of the 8-kb genomic DNA fragment that complements the syrA phenotype revealed high conservation among a broad spectrum of fluorescent pseudomonads . The putative protein encoded by open reading frame 4 (ORF4) (1,299 bp) in the syrA locus region exhibited 85% identity to ArgA, which is involved in arginine biosynthesis in Pseudomonas aeruginosa . Growth of strain W4S2545, the syrA mutant, required supplementation of N minimal medium with arginine . Similarly, syringomycin production of syrA mutant W4S2545 was restored by the addition of arginine to culture media . Furthermore, the insertion of Tn5 in the genome of the syrA mutant W4S2545 was localized between nucleotides 146 and 147 in ORF4, and syringomycin production was complemented in trans with the wild-type DNA fragment containing intact ORF4 . These results demonstrate that the syrA locus is the argA gene of P . syringae pv . syringae and that argA is directly involved in arginine biosynthesis and therefore indirectly affects syringomycin production because of arginine deficiency.

Int J Antimicrob Agents, 2003 Dec, 22(6), 574 - 8
Pharmacodynamics of piperacillin in severely ill patients evaluated by using a PK/PD model; Sauermann R et al.; Penetration of antiinfective drugs into soft tissues is essential for antimicrobial killing at the target site, but is substantially lower in severely ill patients compared with healthy subjects . The present study was conducted to assess the antimicrobial effect of piperacillin in severely ill patients . Strains of Staphylococcus aureus and Pseudomonas aeruginosa were exposed in vitro to concentrations of piperacillin, simulating the pharmacokinetic profiles measured in soft tissue of patients and healthy subjects . The simulation for patients resulted in effective killing, whereas bacterial regrowth was detected for healthy subjects . Our in vitro simulation showed that bacterial killing may be effective in severely ill patients despite relatively low concentrations of piperacillin at the target site . This finding is due to impaired renal function and subsequently prolonged tissue and plasma half-lives of piperacillin in intensive care patients.

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