J Antimicrob Chemother, 2000 Dec, 46(6), 901 - 4 Combined effects of meropenem and aminoglycosides on Pseudomonas aeruginosa in vitro; Nakamura A et al.; To investigate combinations of antibiotics against Pseudomonas aeruginosa, the in vitro effects of combinations of meropenem with each of three aminoglycosides, arbekacin, amikacin and netilmicin, were evaluated using an agar dilution chequerboard technique . The combinations of meropenem and aminoglycosides were effective against almost all P . aeruginosa strains tested, which included meropenem-resistant strains . Increased synergic effects were observed in combinations that included arbekacin or amikacin . None of the combinations had an antagonistic effect . Most of the synergic and additive effects were achieved at clinically relevant concentrations. J Antimicrob Chemother, 2000 Dec, 46(6), 885 - 93 Influence of the MexA-MexB-oprM multidrug efflux system on expression of the MexC-MexD-oprJ and MexE-MexF-oprN multidrug efflux systems in Pseudomonas aeruginosa; Li XZ et al.; Of the Pseudomonas aeruginosa multidrug efflux systems, MexAB-OprM is expressed in wild-type cells, while MexCD-OprJ is not, and MexEF-OprN shows variable, strain-specific expression . In defined mutant strains, MexCD-OprJ expression increased with decreases in MexAB-OprM and was generally inversely related to MexAB-OprM expression . In so-called wild-type strains expressing MexEF-OprN, MexAB-OprM hyperexpression correlated with a decline in MexEF-OprN expression, while loss of MexAB-OprM was associated with increased expression of MexEF-OprN, also indicative of an inverse correlation between MexAB-OprM and MexEF-OprN expression . Still, the increases in MexCD-OprJ and MexEF-OprN failed to compensate for the loss of MexAB-OprM with respect to antibiotic resistance . Nonetheless, these data suggest that the overall complement of these MDR efflux systems is monitored and that alterations in the level of one efflux system may effect compensatory changes in the levels of the others. J Clin Microbiol, 2000 Dec, 38(12), 4614 - 5 Evaluation of random amplified polymorphic DNA typing of Pseudomonas aeruginosa; Campbell M et al.; A method for distinguishing among Pseudomonas aeruginosa strains using random amplified polymorphic DNA (RAPD) typing was evaluated for reproducibility and discriminatory power . A total of 200 isolates, blinded in triplicate, were evaluated by RAPD . All 600 samples were typeable; 197 of 200 isolates gave identical results on all three occasions, and 131 distinct RAPD types were identified. J Clin Microbiol, 2000 Dec, 38(12), 4445 - 52 Simple and inexpensive but highly discriminating method for computer-assisted DNA fingerprinting of Pseudomonas aeruginosa; Al-Samarrai TH et al.; We describe here a method for computer-assisted fingerprinting of Pseudomonas aeruginosa . In this method, DNA is digested with SalI, and bands with molecular sizes of >/=9.7 kb are visually scored after electrophoresis on agarose gels . Pattern scores are entered into a Microsoft Excel database . In scoring, the number of bands within each of a set of molecular size ranges is scored, rather than the absolute molecular size of each band, substantially enhancing the speed and reproducibility of the method, while eliminating the need for using expensive gel scanning equipment and software . Pattern scores are used to generate matrices of genetic distance values, which can be visualized in neighbor-joining trees . The method reliably distinguishes two epidemiologically unrelated isolates in 99.3% of all comparisons . The genetic relationships between isolates observed with the method were consistent with those obtained by analysis of two P . aeruginosa genes, indicating that it provides valid estimates of genetic divergence between isolates . Using the method, respiratory tract isolates from cystic fibrosis patients in Green Lane Hospital in Auckland, New Zealand, were shown to be genetically less diverse than epidemiologically unrelated isolates from other patients . This finding was not due to the existence of clusters of related strains specialized toward colonization of the respiratory tract and thus was indicative of transmission between patients . Analysis of multiple isolates from individual cystic fibrosis patients suggested that up to five separate clusters of genetically related strains may simultaneously be present in a patient . The method described should significantly enhance our ability to investigate the epidemiology of P . aeruginosa. Biochemistry, 2000 Dec 5, 39(48), 14847 - 64 Interaction of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: conformation of the bound peptide; Campbell AP et al.; The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P . aeruginosa infections . We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein . The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond . The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) . The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135) . These turns have been implicated in cross-reactive antibody recognition . (15)N-edited NMR spectroscopy was used to study the binding of the (15)N-labeled PAK pilin peptide to an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus . The results of these studies are as follows: the trans and cis isomers bind with similar affinity to the Fab, despite their different topologies; both isomers maintain the conformational integrity of their beta-turns when bound; binding leads to the preferential stabilization of the first turn over the second turn in each isomer; and binding leads to the perturbation of resonances within regions of the trans and cis backbone that undergo microsecond to millisecond motions . These slow motions may play a role in induced fit binding of the first turn to Fab PAK-13, which would allow the same antibody combining site to accommodate either trans or cis topology . More importantly for vaccine design, these motions may also play a role in the development of a broad-spectrum vaccine capable of generating an antibody therapeutic effective against the multiple strains of P . aeruginosa. Mol Biotechnol, 2000 Sep, 16(1), 5 - 16 Substitution of Glu-59 by Val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme; Karmali A et al.; A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase . The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene . The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography . The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method . The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70-80% . The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles . The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography . On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M(r) of 38,000 and 78,000 Dalton, respectively . These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme . Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase. Ann Pharmacother, 2000 Nov, 34(11), 1238 - 42 Safety and tolerability of bolus intravenous colistin in acute respiratory exacerbations in adults with cystic fibrosis; Conway SP et al.; OBJECTIVE: To assess the safety and tolerability of bolus intravenous doses of colistin during acute respiratory exacerbations in adults with cystic fibrosis and chronic Pseudomonas aeruginosa infection . METHODS: Twelve patients with acute exacerbations of cystic fibrosis were enrolled in a Phase I open-label study . On day 1, patients received three doses of colistin 2 mega-units (160 mg), reconstituted in 50 mL of NaCl 0.9%, by infusion over 30 minutes three times daily . On days 2, 3, and 4, the same dose of colistin was administered by bolus injection three times a day over five minutes after reconstitution in 20, 15, and 10 mL of NaCl 0.9%, respectively . The injection was given by a nurse or physician using a hand-held syringe . If the latter dose was tolerated, it was continued for the remaining eight days of the study . If any dose was not tolerated, treatment reverted to the previously tolerated concentration, which was continued throughout the remainder of the study . RESULTS: No serious adverse events occurred during the course of the trial . Patients without total indwelling venous access systems experienced mild to moderate injection pain . There were no clinically significant changes in renal function . CONCLUSIONS: This study indicates that the administration of bolus intravenous colistin as 2 mega-units (160 mg) in 10 mL of NaCl 0.9% three times a day is safe . It is well-tolerated by patients with total indwelling venous access systems. Appl Environ Microbiol, 2000 Dec, 66(12), 5206 - 12 Influence of infected cell growth state on bacteriophage reactivation levels; Kadavy DR et al.; Reactivation of UV-C-inactivated Pseudomonas aeruginosa bacteriophages D3C3, F116, G101, and UNL-1 was quantified in host cells infected during the exponential phase, during the stationary phase, and after starvation (1 day, 1 and 5 weeks) under conditions designed to detect dark repair and photoreactivation . Our experiments revealed that while the photoreactivation capacity of stationary-phase or starved cells remained about the same as that of exponential-phase cells, in some cases their capacity to support dark repair of UV-inactivated bacteriophages increased over 10-fold . This enhanced reactivation capacity was correlated with the ca . 30-fold-greater UV-C resistance of P . aeruginosa host cells that were in the stationary phase or exposed to starvation conditions prior to irradiation . The dark repair capacity of P . aeruginosa cells that were infected while they were starved for prolonged periods depended on the bacteriophage examined . For bacteriophage D3C3 this dark repair capacity declined with prolonged starvation, while for bacteriophage G101 the dark repair capacity continued to increase when cells were starved for 24 h or 1 week prior to infection . For G101, the reactivation potentials were 16-, 18-, 10-, and 3-fold at starvation intervals of 1 day, 1 week, 5 weeks, and 1 . 5 years, respectively . Exclusive use of exponential-phase cells to quantify bacteriophage reactivation should detect only a fraction of the true phage reactivation potential. Clin Cardiol, 2000 Nov, 23(11), 808 - 10 Coronary artery stent infection; Dieter RS; This paper aimed to examine the literature for cases of coronary artery stent infection in order to provide comprehensive data to clinicians regarding its prevalence, clinical presentations, and possible treatments . Coronary artery stenting was initially reported in 1987 . Stenting of the coronary arteries is now used in 40-60% of all interventional coronary artery procedures . The understanding of the pathophysiology of coronary artery disease is evolving . It has been suggested that atherosclerosis may be a complication of an infectious etiology . By using a stent to treat coronary artery disease, a foreign body is directly juxtaposed with an area of inflammation . The first reported case of an infected coronary artery stent was in 1993 . Although this is an exceedingly rare event, the associated mortality is alarmingly high . Analysis of the literature reveals a total of four reported cases of coronary artery stent infection . Symptoms of stent infection present days to weeks after the initial coronary intervention . All four patients developed fevers and at least two patients developed postintervention angina . In patients who have had a coronary artery stented, the presence of angina and fevers should make the clinician suspicious for a stent-related infection . Two of the patients had infection with Pseudomonas aeruginosa, which seems to be an unusual organism for a catheter-related infection . Surgical removal of the infected stent and artery complex was performed on nearly all cases . Despite aggressive measures, the majority of patients died . Few data are available on the long-term risk for coronary artery stent infection . In a patient who has undergone coronary artery stent placement, the clinician must be very sensitive to fever, return of angina, and bacteremia . The complication rate at the present time does not warrant the use of prophylactic antibiotics prior to high-risk procedures (e.g., dental procedures) . Furthermore, the low infection rate of coronary artery stents may be a result of the inflammatory nature of atherosclerosis, which may provide a protective benefit against bacterial infection of the stent. Clin Infect Dis, 2000 Dec, 31(6), 1349 - 56 Epub 2000 Nov 29. Pseudomonas aeruginosa community-acquired pneumonia in previously healthy adults: case report and review of the literature; Hatchette TF et al.; We report a case of rapidly fatal Pseudomonas aeruginosa community-acquired pneumonia (CAP) in a previously healthy 67-year-old woman . Eleven published case reports of P . aeruginosa CAP in previously healthy adults are reviewed . According to our review, the mean age of affected patients is 45.3 years . Five patients described in the literature were smokers with a mean smoking history of 40 pack-years . The clinical presentation is nonspecific, and although the pneumonia can be rapidly fatal, only 33% of the patients who were reported died . However, mortality may be independent of treatment within the first 36 hours of presentation . Exposure to aerosols of contaminated water is a risk factor for P . aeruginosa CAP in this population . Pseudomonas CAP should be considered in the differential diagnosis for anyone with a smoking history who presents with rapidly progressive pneumonia . We discuss treatment recommendations that are based on evidence in the currently available literature on the subject. Clin Infect Dis, 2000 Dec, 31(6), 1331 - 7 Epub 2000 Nov 29. Outbreak of severe Pseudomonas aeruginosa infections caused by a contaminated drain in a whirlpool bathtub; Berrouane YF et al.; During a 14-month period, 7 patients with hematological malignancies acquired serious infections caused by a single strain of multiply resistant Pseudomonas aeruginosa . A case-control study, culture surveys, and pulsed-field gel electrophoresis implicated a whirlpool bathtub on the unit as the reservoir . All case patients and 32% of control patients used this bathtub (P=.003) . The epidemic strain was found only in cultures of samples taken from the bathtub . The drain of the whirlpool bathtub, which was contaminated with the epidemic strain, closed approximately 2.54 cm below the drain's strainer . Water from the faucet, which was not contaminated, became contaminated with P . aeruginosa from the drain when the tub was filled . The design of the drain allowed the epidemic strain to be transmitted to immunocompromised patients who used the whirlpool bathtub . Such tubs are used in many hospitals, and they may be an unrecognized source of nosocomial infections . This potential source of infection could be eliminated by using whirlpool bathtubs with drains that seal at the top. Microb Pathog, 2000 Dec, 29(6), 345 - 56 Pseudomonas aeruginosa induces apoptosis in human endothelial cells; Valente E et al.; Pseudomonas aeruginosa has been shown to enter into human endothelial cells in vitro . To ascertain the effects of bacterial intracellular (IC) infection, endothelial cells were exposed to PAK and PAO-1 strains for 1 h and treated with gentamicin in culture medium for different periods . P . aeruginosa induced a significant production of superoxide and hydrogen peroxide by endothelial cells . Concentrations of IC bacteria were reduced progressively with time and no viable PAO-1 was detected at 24 h after infection . However, IC infection led to killing of 32.2%+/-2.9 and 51.8%+/-3.5 of the cells infected with PAK and PAO-1, respectively, as determined by the MTT assay . By three criteria (transmission electron microscopy, DNA electrophoresis and reactivity with annexin V) infected cells exhibited features of apoptosis . Treatment of infected cells with anti-oxidants (catalase, tocopherol and N -acetyl-L-cysteine) significantly decreased the percentage of cell death . In contrast, treatment with aminoguanidine, an inhibitor of inducible NO synthase, increased significantly the killing of PAO-1 infected cells . Based on these results we speculate that in response to P . aeruginosa infection, endothelial cells increase the production of reactive oxygen intermediates to eliminate IC pathogens, but cells do not resist the oxidative stress and die by apoptosis . Microb Pathog, 2000 Dec, 29(6), 329 - 43 Triggering the ExoS regulon of Pseudomonas aeruginosa: A GFP-reporter analysis of exoenzyme (Exo) S, ExoT and ExoU synthesis; Hornef MW et al.; The ExoS regulon of Pseudomonas aeruginosa encodes diverse type III secreted effector proteins which have been shown to exert cytotoxic effects in cell culture experiments . However, little information exists about the environmental conditions and stimuli for upregulation of the ExoS regulon . Translational reporter fusion proteins of exoenzyme (Exo) S, ExoT and ExoU, as well as the type II secreted exotoxin A (ETA) to the green fluorescent protein (GFP), were constructed in order to compare exoprotein production under diverse growth conditions . Reporter protein activity was recorded by FACS-analysis and by conventional and confocal laser scanning microscopy . Low ion concentration induced co-ordinated upregulation of ExoS, ExoT and ExoU with a maximum effect at 37 degrees C . A dose-dependent upregulation was seen with human serum or increasing NaCl concentrations . A type III secretion-negative pcrD mutant of P . aeruginosa showed a weak ExoS response to environmental stimuli, compared with the parental strain, suggesting a negative regulatory mechanism . Co-culture with the mammalian cell lines J774A.1 or HeLa led to rapid upregulation of ExoS, ExoT and ExoU synthesis . These data suggest that the ExoS regulon of P . aeruginosa can be triggered by a variety of environmental signals as well as by cell contact with eukaryotic cells . Invest Ophthalmol Vis Sci, 2000 Dec, 41(13), 4189 - 94 Membrane-type matrix metalloproteinases in mice intracorneally infected with Pseudomonas aeruginosa; Dong Z et al.; PURPOSE: To establish the presence of membrane-type matrix metalloproteinases (MT-MMPs) in the cornea and their expression in naive and immunized mice intracorneally infected with Pseudomonas aeruginosa . METHODS: Naive (unimmunized) and immunized C57BL/6J mice were infected with P . aeruginosa, and gene expression of MT-MMPs were detected by RT-PCR . Immunoblot analysis and immunostaining were also used to characterize the MT-MMP response in both sets of animals . RESULTS: Expression of MT1-MMP, MT2-MMP, and MT3-MMP (MMP 14, 15, and 16) was detected by RT-PCR and immunoblot analysis . Of the three MT-MMPs detected, MT1-MMP exhibited the greatest expression at protein levels . In general, a bell-shaped curve was obtained for each of the MT-MMPs in naive mice, but all of them showed much less expression in the immunized mice . MT1-MMP was localized in the epithelial tissue of the cornea, whereas MT2-MMP and MT3-MMP were mainly found in the interface between the epithelium and substantia propria . CONCLUSIONS: MT1-MMP was detected and expressed to a greater extent in naive mice than MT2-MMP and MT3-MMP . Peak expression of all three MT-MMPs showed a good correlation with the overall inflammatory response. Invest Ophthalmol Vis Sci, 2000 Dec, 41(13), 4080 - 4 C57BL/6 mice lacking Muc1 show no ocular surface phenotype; Danjo Y et al.; PURPOSE: To test the hypothesis that a membrane-spanning mucin, Muc1, facilitates the spread of tear film and protects against bacterial adherence . METHODS: Age-matched, Muc1 null mice and wild-type mice of C57BL/6 genetic background were used for comparison . Eyes were examined by slit lamp biomicroscopy with fluorescein solution to assess epithelial damage and tear film stability . Structure of the ocular surface epithelia was examined by light microscopy, scanning and transmission electron microscopy, and wholemount confocal microscopy . Bacterial adherence assay was performed on in vivo corneas with Pseudomonas aeruginosa containing a plasmid encoding green fluorescent protein, followed by wholemount confocal microscopy . Real-time reverse transcription-polymerase chain reaction was performed using Muc4-specific primers to quantitate Muc4 mRNA expression in ocular surface tissues . RESULTS: No differences were found between Muc1 null and control mice in any parameter tested . Ocular surface epithelia of Muc1 null mice of the C57BL/6 strain had a normal appearance of surface microplicae, a well-developed glycocalyx on the apical cell membrane, and a normal appearance of goblet cell mucin packets . There was no convincing evidence that bacterial adherence on the cornea was increased in Muc1 null mice . Muc4 mRNA expression was not upregulated in Muc1 null mice compared with control . No ocular surface infections were observed in Muc1 null mice of the C57BL/6 strain (n = 204), which were housed in the animal facility over a period of 26 months . CONCLUSIONS: Muc1 null mice of C57BL/6 background appeared normal in all respects tested . These data differ from the reported phenotype in the mice of the C57BL/6 x SVJ129 background, which show development of blepharitis and conjunctivitis. Cornea, 2000 Nov, 19(6), 777 - 81 Pathogenesis and treatment of "sterile" midperipheral corneal infiltrates associated with soft contact lens use; Baum J et al.; PURPOSE: To demonstrate the sterile nature of presumed sterile midperipheral corneal infiltrates associated with soft contact lens (SCL) use and to show that withholding antibiotics or the occasional use of a topical corticosteroid alone may, with strict guidelines, have a role in the treatment of this entity . METHODS: Nine consecutive patients presenting with typical midperipheral corneal infiltrates after SCL wear were seen in the office (O.H.D.) during a 2-year period, 1996-1998 . All patients were initially placed on topical fluorometholone as the only treatment . RESULTS: Eight of the nine patients experienced a rapid relief of symptoms and the infiltrates were noted to be smaller and less dense in 34 days . Therapy was discontinued after 7 days, by which time the lesions had cleared . The ninth patient developed a microbial keratitis from which Pseudomonas aeruginosa was cultured . With appropriate therapy, visual acuity returned to 20/25 . Two different algorithms are offered for the treatment of a putative sterile infiltrate associated with SCL use . CONCLUSION: The use of a topical corticosteroid alone may have a role in the treatment of presumed sterile midperipheral corneal infiltrates associated with SCLs when strict guidelines are followed . Such therapy suggests that the infiltrates are not the result of infection. Genetika, 2000 Oct, 36(10), 1330 - 9 {Properties of natural interspecific hybrids of transposable phages of Pseudomonas aeruginosa: specific characteristics of phage PL24 transposition}; Mit'kina LN et al.; Properties of natural hybrid transposable phages (TP) of Pseudomonas aeruginosa, including phage PL24 and lysogens for this phage, were studied . PL24 possesses the properties of TP from two previously described groups, B3 and D3112 . Its genome, unlike the genome of D3112, contains many sites susceptible to the SalGI restriction endonuclease and possesses no more than 100 nucleotides of bacterial origin located at the left genome end . However, unlike B3, phage PL24 failed to induce auxotrophic mutants upon integration in the bacterial genome . This phage differed from both B3 and D3112 in sensitivity to chloroform treatment . A more detailed examination of a group containing 25 randomly isolated lysogens for phage PL24 revealed previously unknown processes occurring at early stages of bacterial lysogenization . There are at least two different modes of cell lysogenization with phage PL24 . In the first case, the emerging lysogens contained a single prophage genome located (in each lysogen) at individual sites . In the second case, polylysogenic bacteria appeared, and, after primary integration of a phage genome, replicative transposition occurred at new sites (often accompanied by the appearance of prophage clusters at these sites) . The choice of the mode of lysogenization can be determined both by differences in the physiological state of bacteria and by specific features of phage PL24, which possibly affect the time of repressor accumulation to the concentration sufficient for blocking phage growth or the stability of the lysogenic state. Braz J Infect Dis, 1999 Jun, 3(3), 97 - 110 Bacterial Pathogens Isolated from Patients with Bloodstream Infections in Latin America, 1997: Frequency of Occurrence and Antimicrobial Susceptibility Patterns from the SENTRY Antimicrobial Surveillance Program; Sader HS et al.; We report the antimicrobial susceptibility of 736 organisms isolated from bloodstream infections in 10 Latin American medical centers during the first six months of 1997 . The data presented here is from the SENTRY Antimicrobial Surveillance Program, a comprehensive surveillance study involving 72 medical centers worldwide . The isolates ivere tested for in in vitro susceptibility to 35 antimicrobial agents by the broth microdilution method . The five most frequently isolated species were (n/%): Staphylococcus aureus (1 65/22.4%), Escherichia coli(118/16.0%), coagulase-negative staphylococci (CoNS - 115/15.6%), Pseudomonas aeruginosa (51/6.9%), Klebsiella spp . (46/ 6.3%) . Susceptibility to oxacillin was 70.9% for S . aureus and only 33.9% for CoNS . Vancomycin was active against all of staphylococci, while teicoplanin was active against 99.4% of S . aureus and only 90.4% of CoNS . The new fluoroquinolones sparfloxacin, gatifloxacin, and trovafloxacin, and the streptogramin, quinupristin/dalfopristin, were very active against these species . Only one vancomycin-resistant enterococcus was detected; however, high-level aminoglycoside resistance rates were common (66.7%) . E . coli and Klebsiella spp . showed low susceptibilities for cefotaxime (90.7% and 41.3%) and for cefoxitin (85.6% and 78.3% respectively), indicating a high frequency of isolates that produce ESBL and/or stably derepressed ampC enzymes . These strains, phenotypically consistent with extended-spectrum beta-lactamase (ESBL) production, were typed using ribotyping and pulsed-field gel electrophoresis . The most active compounds (M IC90 in microg/mL /% susceptibility) against P . aeruginosa were meropenem (2 /94.1%), followed by amikacin (>32 / 86.3%), and piperacillin alone or with tazobactam (128/84.3%) . Ceftazidime and cefepime showed similar activity (70.6% susceptibility) and levofloxacin was the most active fluoroquinolone (MI C50 &lte; 0.5; 76.5% susceptibility) against this gram-negative species . These results show the unique pattern of bloodstream isolates for Latin America and they demonstrate the present utility of several classes of compounds against emerging antimicrobial-resistant species in this region. Curr Infect Dis Rep, 2000 Dec, 2(6), 490 - 496 Waterborne Nosocomial Infections; Squier C et al.; Waterborne pathogens cause infections in health-care facilities . Despite guidelines addressing these pathogens, outbreaks and pseudo-outbreaks continue to occur . We reviewed recent reports of infections caused by Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Chryseobacterium species, nontuberculous mycobacteria, and Legionella species . Mycobacterium avium complex (MAC) infection in HIV patients has been linked to hospital water distribution systems; molecular subtyping showed that MAC isolates in patients and hospital water were identical . In immunosuppressed patients, Fusarium infection has been linked to the hospital water distribution system; again molecular subtyping showed that isolates from patients and the water supply were identical . Parasites, especially Cryptosporidium, and viruses have also been implicated in nosocomial infection . Transmission occurs via contact, ingestion, aspiration, or aerosolization of potable water, or via the hands of health-care workers . Interventions designed to interrupt transmission of waterborne pathogens have included the use of antimicrobial handwashes, targeted disinfection of the water supply, and, in high-risk populations, restricting the use of tap water. Curr Infect Dis Rep, 1999 Oct, 1(4), 338 - 346 The Red Menace: Emerging Issues in Antimicrobial Resistance in Gram-Negative Bacilli; Rice LB et al.; Gram-negative bacilli cause more than one third of all nosocomial infections in US hospitals . Despite a surfeit of new and highly potent antimicrobial agents, the problem of resistance in these pathogens continues to increase . Particularly important is the emergence of resistance to the fluoroquinolone and beta-lactam classes of antimicrobial agents . Recent work has confirmed that resistance to fluoroquinolone antibiotics is a complex process that involves mutations in the target enzymes (topoisomerase II and IV), decreased access to the target enzyme resulting from low permeability of the outer membrane (this is primarily important in Pseudomonas aeruginosa), and active efflux from the cell . Resistance to beta-lactam antibiotics, however, is primarily caused by the elaboration of an ever-growing number of beta-lactamases . Our ability to understand the genetic and biochemical underpinnings of these resistance phenotypes will be an important factor in determining the ultimate success of efforts to control their emergence and spread. J Bacteriol, 2000 Dec, 182(24), 7070 - 4 High-frequency flp recombinase-mediated inversions of the oriC-containing region of the Pseudomonas aeruginosa genome; Barekzi N et al.; The genomes of the two clonally derived Pseudomonas aeruginosa prototypic strains PAO1 and DSM-1707 differ by the presence of a 2 . 19-Mb inversion including oriC . Integration of two Flp recombinase target sites near the rrn operons containing the inversion endpoints in PAO1 led to Flp-catalyzed inversion of the intervening 1.59-Mb fragment, including oriC, at high frequencies (83%), favoring the chromosome configuration found in DSM-1707 . The results indicate that the oriC-containing region of the P . aeruginosa chromosome can readily undergo and tolerate large inversions. J Bacteriol, 2000 Dec, 182(24), 6999 - 7006 Proteome analysis of the effect of mucoid conversion on global protein expression in Pseudomonas aeruginosa strain PAO1 shows induction of the disulfide bond isomerase, dsbA; Malhotra S et al.; Pseudomonas aeruginosa strains that cause chronic pulmonary infections in cystic fibrosis patients typically undergo mucoid conversion . The mucoid phenotype indicates alginate overproduction and is often due to defects in MucA, an antisigma factor that controls the activity of sigma-22 (AlgT {also called AlgU}), which is required for the activation of genes for alginate biosynthesis . In this study we hypothesized that mucoid conversion may be part of a larger response that activates genes other than those for alginate synthesis . To address this, a two-dimensional (2-D) gel analysis was employed to compare total proteins in strain PAO1 to those of its mucA22 derivative, PDO300, in order to identify protein levels enhanced by mucoid conversion . Six proteins that were clearly more abundant in the mucoid strain were observed . The amino termini of such proteins were determined and used to identify the gene products in the genomic database . Proteins involved in alginate biosynthesis were expected among these, and two (AlgA and AlgD) were identified . This result verified that the 2-D gel approach could identify gene products under sigma-22 control and upregulated by mucA mutation . Two other protein spots were also clearly upregulated in the mucA22 background, and these were identified as porin F (an outer membrane protein) and a homologue of DsbA (a disulfide bond isomerase) . Single-copy gene fusions were constructed to test whether these proteins were enhanced in the mucoid strain due to increased transcription . The oprF-lacZ fusion showed little difference in levels of expression in the two strains . However, the dsbA-lacZ fusion showed two- to threefold higher expression in PDO300 than in PAO1, suggesting that its promoter was upregulated by the deregulation of sigma-22 activity . A dsbA-null mutant was constructed in PAO1 and shown to have defects predicted for a cell with reduced disulfide bond isomerase activity, namely, reduction in periplasmic alkaline phosphatase activity, increased sensitivity to dithiothreitol, reduced type IV pilin-mediated twitching motility, and reduced accumulation of extracellular proteases, including elastase . Although efficient secretion of elastase in the dsbA mutant was still demonstrable, the elastase produced appeared to be unstable, possibly as a result of mispaired disulfide bonds . Disruption of dsbA in the mucoid PDO300 background did not affect alginate production . Thus, even though dsbA is coregulated with mucoid conversion, it was not required for alginate production . This suggests that mucA mutation, which deregulates sigma-22, results in a global response that includes other factors in addition to increasing the production of alginate. J Bacteriol, 2000 Dec, 182(24), 6940 - 9 Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa; Pessi G et al.; Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN) . This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase . The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P . aeruginosa PAO1 . The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR . Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp . Their function was confirmed by transcriptional lacZ fusions . The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1 . Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides . The lux box was recognized by both LasR {activated by N-(oxododecanoyl)-homoserine lactone} and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P . aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P . fluorescens CHA0 . A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression . Without LasR and RhlR, ANR could not activate the hcn promoter . Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone . Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR. Intensive Care Med, 2000 Sep, 26(9), 1386 - 9 Endogenous Pseudomonas aeruginosa endophthalmitis: a case report and literature review; Reedy JS et al.; Endogenous endophthalmitis is a vision-threatening condition that results from the hematogenous spread of infection to the eye that originated in a distant primary focus . Although it has long been recognized that bloodborne organisms can infect the eye, endogenous bacterial endophthalmitis is considered a rare entity . We present a unique case of a critically ill patient with a cholangiocarcinoma complicated by ascending cholangitis who developed endogenous Pseudomonas aeruginosa endophthalmitis . An awareness of the risk factors predisposing to endogenous endophthalmitis and a high clinical suspicion are necessary to make an early diagnosis in the intensive care unit . Management involves an aggressive combined medical and surgical approach in an effort to prevent ocular morbidity and vision loss. Biochemistry, 2000 Nov 28, 39(47), 14504 - 14 Interaction of polyphemusin I and structural analogs with bacterial membranes, lipopolysaccharide, and lipid monolayers; Zhang L et al.; Three structural variants (PV5, PV7, and PV8) of the horseshoe crab cationic antimicrobial peptide polyphemusin I were designed with improved amphipathic profiles . Circular dichroism spectroscopy analysis indicated that in phosphate buffer polyphemusin I, PV7, and PV8 displayed the spectrum of a type II beta-turn-rich structure, but, like polyphemusin I, all three variants adopted a typical beta-sheet structure in an anionic lipid environment . Both polyphemusin I and variants were potent broad spectrum antimicrobials that were clearly bactericidal at their minimal inhibitory concentrations . The variants were moderately less active in vitro but more effective in animal models . Moreover, these variants exhibited delayed bacterial killing, whereas polyphemusin I killed Escherichia coli UB1005 within 5 min at 2.5 microg/mL . All the peptides showed similar abilities to bind to bacterial lipopolysaccharide (LPS) and permeabilize bacterial outer membranes . Consistent with this was the observation that all peptides significantly inhibited cytokine production by LPS-stimulated macrophages and penetrated polyanionic LPS monolayers to similar extents . None of the peptides had affinity for neutral lipids as evident from both tryptophan fluorescence spectroscopy and Langmuir monolayer analysis . As compared to polyphemusin I, all variants showed reduced ability to interact with anionic lipids, and the hemolytic activity of the variants was decreased by 2-4-fold . In contrast, polyphemusin I efficiently depolarized the cytoplasmic membrane of E . coli, as assessed using a membrane potential sensitive fluorescent dye 3,3-dipropylthiacarbocyanine (diSC(3)5) assay, but the variants showed a substantially delayed and decreased depolarizing ability . The coincident assessment of cell viability indicated that depolarization of the bacterial cytoplasmic membrane potential by polyphemusin I occurred prior to lethal damage to cells . Our data suggest that increase of amphipathicity of beta-sheet polyphemusin I generally resulted in variants with decreased activity for membranes . Interestingly, all variants showed an improved ability to protect mice both against infection by Pseudomonas aeruginosa and from endotoxaemia. J Trauma, 2000 Nov, 49(5), 873 - 8 Locally delivered antibodies combined with systemic antibiotics confer synergistic protection against antibiotic-resistant burn wound infection; Felts AG et al.; BACKGROUND: Nosocomially derived gram-negative infections, particularly from antibiotic-resistant pathogens, are a cause of morbidity in patients with severe burn wounds . METHODS: Locally delivered polyclonal antibodies and systemically infused ceftazidime were combined in a lethal murine burn wound model against a virulent Pseudomonas aeruginosa strain that exhibits intermediate resistance to ceftazidime . RESULTS: Survival was synergistically enhanced in cohorts of burned mice treated both locally (subeschar) with pooled polyclonal human immunoglobulin G (1-mg dose) and intravenously with infused ceftazidime (0.44 mg dose) . Enhancement of survival correlated with reduced bacterial quantitation in local and systemic tissue observed in separate burned cohorts . Burned, infected mice treated prophylactically with either individual treatment at the same dose or a combination of both treatments administered systemically showed no survival enhancement as compared with the untreated control group . CONCLUSION: Treatment of antibiotic-resistant burn wound infections with antibiotics together with locally delivered immunoglobulins may improve antibiotic protective effects against antibiotic-resistant pathogens. Rev Esp Quimioter, 2000 Sep, 13(3), 281 - 5 {Risk factors and prognostics of nosocomial infectionof surgical wounds in a general hospital}; Martinez B et al.; Surgical infections due to gram-negative bacteria are important because of their high frequency, morbidity and mortality . In order to evaluate the risk factors and prognostics of gram-negative surgical wound infections a group of 50 patients with surgical infections were studied prospectively and consecutively and were compared with another group of 50 patients with similar characteristics but no infection . No significant differences were observed with respect to age between the two groups . Previous surgery, prior surgical infections and use of wide-spectrum antibiotics in the six weeks before the study were significantly associated with the development of surgical wound infections due to gram-negative bacteria . The most isolated bacteria were Pseudomonas aeruginosa (34%), followed by polymicrobial flora (16%) . The factors significantly associated with a poor prognosis were the following: severe underlying disease, a clinically critical situation, previous surgery, arterial hypertension, complications, type of gram-negative bacteria, prior use of wide-spectrum antibiotics in the previous six weeks and older age . No deaths occurred. J Immunol, 2000 Dec 1, 165(11), 6496 - 503 Intrapulmonary TNF gene therapy reverses sepsis-induced suppression of lung antibacterial host defense; Chen GH et al.; Sepsis syndrome is frequently complicated by the development of nosocomial infections, particularly Gram-negative pneumonia . Although TNF-alpha (TNF) has been shown to mediate many of the pathophysiologic events in sepsis, this cytokine is a critical component of innate immune response within the lung . Therefore, we hypothesized that the transient transgenic expression of TNF within the lung during the postseptic period could augment host immunity against nosocomial pathogens . To test this, mice underwent 26-gauge cecal ligation and puncture (CLP) as a model of abdominal sepsis, followed 24 h later by intratracheal (i.t.) administration of PSEUDOMONAS: aeruginosa . In animals undergoing sham surgery followed by bacterial challenge, PSEUDOMONAS: were nearly completely cleared from the lungs by 24 h . In contrast, mice undergoing CLP were unable to clear P . aeruginosa and rapidly developed bacteremia . Alveolar macrophages (AM) recovered from mice 24 h after CLP produced significantly less TNF ex vivo, as compared with AM from sham animals . Furthermore, the adenoviral mediated transgenic expression of TNF within the lung increased survival in CLP animals challenged with PSEUDOMONAS: from 25% in animals receiving control vector to 91% in animals administered recombinant murine TNF adenoviral vector . Improved survival in recombinant murine TNF adenoviral vector-treated mice was associated with enhanced lung bacterial clearance and proinflammatory cytokine expression, as well as enhanced AM phagocytic activity and cytokine expression when cultured ex vivo . These observations suggest that intrapulmonary immunostimulation with TNF can reverse sepsis-induced impairment in antibacterial host defense. J Biol Inorg Chem, 2000 Oct, 5(5), 551 - 9 Copper coordination in blue proteins; Gray HB et al.; The spectroscopic and electrochemical properties of blue copper proteins are strikingly different from those of inorganic copper complexes in aqueous solution . Over three decades ago this unusual behavior was ascribed to constrained coordination in the folded protein; consistent with this view, crystal structure determinations of blue proteins have demonstrated that the ligand positions are essentially unchanged on reduction as well as in the apoprotein . Blue copper reduction potentials are tuned to match the particular function of a given protein by exclusion of water from the metal site and strict control of the positions of axial ligands in the folded structure . Extensive experimental work has established that the reorganization energy of a prototypal protein, Pseudomonas aeruginosa azurin, is approximately 0.7 eV, a value that is much lower than those of inorganic copper complexes in aqueous solution . The lowered reorganization energy in the protein, which is attributable to constrained coordination, is critically important for function, since the driving forces for electron transfer often are low (approximately 0.1 eV) between blue copper centers and distant (>10 A) donors and acceptors. Ann Otolaryngol Chir Cervicofac . 2000 Nov;117(5):291. {Malignant or necrotizing otitis externa: experience in 22 cases}; Martel J et al.; Malignant or progressive necrotizing otitis extrema is an uncommon but severe infectious condition of the external auditory canal . Over a period of four years, we treated 22 patients: 60% had diabetes (1/4 insulin dependent) and 13% were immunodepressed . The causal germ was Pseudomonas aeruginosa in 87% of cases . The pretherapeutic work-up included a computed tomography scan and a technetium scintigraphy to confirm diagnosis and assess extension . Repeated scintigraphies with gallium were used to follow the course under treatment . Medical treatment was used in most cases (16/22) with parenteral antibiotic therapy using a third-generation cephalosporin (ceftazidime or ceftriaxone) and a fluoroquinolone (ciprofloxacin or ofloxacin) and, if there was no contraindication, hyperbaric oxygen . Surgery is not indicated in malignant otitis externa . We had a 95% cure rate with only 10% recurrence . We reviewed the data in the literature on malignant otitis externa and present the important diagnostic, imaging and therapeutic aspects. Braz J Infect Dis, 1999 Dec, 3(6), 231 - 237 Evaluation of the Cephalosporins, Cefepime, Cefpirome and Ceftazidime, against Clinical Isolates of Imipenem-Resistant Pseudomonas aeruginosa; Sader HS et al.; The level of resistance to imipenem in our institution has increased significantly in recent years, particularly in isolates of Pseudomonas aeruginosa . At present, 30% to 40% of P . aeruginosa isolated in clinical samples are imipenem resistant . The objective of this study was to test therapeutic alternatives within the class of beta-lactam antibiotics in the treatment of infections caused by imipenem-resistant strains of P . aeruginosa (IRPA) . We tested 160 isolates of IRPA collected consecutively from in patients at Hospital Sao Paulo-UNIFESP (Federal University of Sao Paulo) . Using the E-test method, these isolates were tested for cefepime, cefpirome, and ceftadizime susceptibility or resistance . The E-test was also used to confirm the resistance to imipenem . One group of samples was studied epidemiologically using the pulsed-field gel electrophoresis (PFGE) method to determine how this type of resistance spread in our institution . Cefepime was the most active antibiotic (MIC50, 24 microg/mL; MIC(90), 64 microg/mL) and only 57 (35.6%) of the samples showed cross-resistance between imipenem and this fourth-generation cephalosporin . The number of samples that were highly resistant (MIC(90), >256 microg/mL) to cefepime, ceftazidime or cefpirome was 8 (5.0%), 22 (13.8%) and 38 (23.8%), respectively . Molecular typing revealed the presence of 9 molecular profiles among 26 IRPA samples tested, suggesting that selection of resistant mutants occurred in each patient . However, identical molecular profiles were also found in more than one patient . This study showed that cefepime is the most effective cephalosporin against the IRPA samples isolated in our institution . In addition, we conclude that P.areuginosa resistant strains are selected in each patient, but patient to patient transmission also occurs. Thorax, 2000 Dec, 55(12), 1033 - 9 Treatment of severe nosocomial pneumonia: a prospective randomised comparison of intravenous ciprofloxacin with imipenem/cilastatin; Torres A et al.; BACKGROUND: A prospective multicentre study was undertaken to compare the efficacy of intravenous ciprofloxacin or imipenem in the treatment of severe nosocomial pneumonia requiring mechanical ventilation . METHODS: Patients with a clinical suspicion of pneumonia were randomised to receive either ciprofloxacin (800-1200 mg/day) or imipenem (2-4 g/day) in doses adjusted for renal function and specimens of the lower respiratory tract were taken . Patients were included in the study when specimens showed significant growth for potentially pathogenic microorganisms in quantitative bacterial cultures (n = 75, ciprofloxacin 41/75 (55%); imipenem 34/75 (45%)) . The clinical and bacteriological success rates were the primary and secondary efficacy variables . An intent-to-treat analysis was performed for all randomised patients who received at least one dose of the study medication (n = 149, ciprofloxacin 72/149 (48%), imipenem 77/149 (52%)) . RESULTS: The success rates were generally good, but neither the clinical success rates (ciprofloxacin, 29/41 (71%), imipenem, 27/34 (79%); 95% CI -10.8 to 28.1; p = 0.435) nor the bacteriological response rate (ciprofloxacin, 20/41 (49%), imipenem, 17/34 (50%); 95% CI -21.5 to 23.9; p = 1.0) were significantly different between the study arms . Pseudomonas aeruginosa was recovered in 26/75 patients (35%) and clinical (ciprofloxacin, 10/14 (71%), imipenem, 8/12 (67%); 95% CI -40.4 to 30.9; p = 1.0) and bacteriological response rates (ciprofloxacin, 7/14 (50%), imipenem, 3/12 (25%), 95% CI -60.9 to 10.9, p = 0.247) were not significantly different in this subgroup of patients . Resistance of Pseudomonas aeruginosa developed in 5/26 cases (19%), 1/14 (7%) to ciprofloxacin and 4/12 (33%) to imipenem (p = 0.147), and the mortality was 12/75 (16%) with no difference between treatment groups (ciprofloxacin, 8/41(24%), imipenem 4/34 (17%); p = 0.362) . The clinical response was evaluable in 109/149 patients (73%) in the intent-to-treat analysis and was successful in 74/109 patients (68%) . The clinical response rates were also not significantly different in the intent-to-treat analysis (ciprofloxacin, 34/52 (65%), imipenem, 40/57 (70%); 95% CI -12.8 to 22.3; p = 0.746) . CONCLUSIONS: Treatment with either ciprofloxacin or imipenem was effective in a selected group of patients with microbiologically confirmed, severe nosocomial pneumonia requiring mechanical ventilation . Although no differences between the study medication could be documented in this trial, smaller differences between treatment arms may have been missed because of sample size limitations. Infect Immun, 2000 Dec, 68(12), 7100 - 13 The arginine finger domain of ExoT contributes to actin cytoskeleton disruption and inhibition of internalization of Pseudomonas aeruginosa by epithelial cells and macrophages; Garrity-Ryan L et al.; Pseudomonas aeruginosa, an important nosocomial pathogen of humans, expresses a type III secretion system that is required for virulence . Previous studies demonstrated that the lung-virulent strain PA103 has the capacity to be either cytotoxic or invasive . Analyses of mutants suggest that PA103 delivers a negative regulator of invasion, or anti-internalization factor, to host cells via a type III secretion system . In this work we show that the type III secreted protein ExoT inhibits the internalization of PA103 by polarized epithelial cells (Madin-Darby canine kidney cells) and J774.1 macrophage-like cells . ExoS, which is closely related to ExoT but has additional ADP-ribosylating activity, can substitute for ExoT as an anti-internalization factor . ExoT contains a signature arginine finger domain found in GTPase-activating proteins . Mutation of the conserved arginine in ExoT diminished its anti-internalization activity and altered its ability to disrupt the actin cytoskeleton . Cell fractionation experiments showed that ExoT is translocated into host cells and that mutation of the arginine finger did not disrupt translocation . In a mouse model of acute pneumonia, PA103DeltaUDeltaT reached the lungs as efficiently as PA103DeltaU but showed reduced colonization of the liver . This finding suggests that the ability to resist internalization may be important for virulence in vivo. Chest, 2000 Nov, 118(5), 1500 - 3 Hemoptysis following left ventricular aneurysm repair: a misleading clinical sign; Kofidis T et al.; We report on a 66-year-old man with severe hemoptysis following coronary artery bypass grafting and repair of a left ventricular septal defect after acute myocardial infarction . Initial diagnosis was delayed by misleading clinical symptoms and radiologic studies . Due to subfebrile temperature and sputum culture positive for Pseudomonas aeruginosa, he had been treated with antibiotics before reoperation . At reoperation, replacement of all foreign material and reconstruction of the ventricular repair with bovine pericardium resulted in reinfection with the same organism despite prolonged antibiotic therapy after 6 months . Removal of the pericardial tissue with direct suture closure of the ventricles and interposition of omentum led to complete healing of the infection without reoccurrence after 2 years. Antimicrob Agents Chemother, 2000 Dec, 44(12), 3322 - 7 Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-oprM efflux pumps in Pseudomonas aeruginosa; Masuda N et al.; To find the exact substrate specificities of three species of tripartite efflux systems of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems . Comparison of the susceptibilities of the constructed mutants to 52 antimicrobial agents belonging to various groups suggested the following substrate specificities . All of the efflux systems extrude a wide variety of antimicrobial agent groups, i.e., quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (all but carbenicillin and sulbenicillin), most cephems (all but cefsulodin and ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin B or imipenem . Extrusion of aminoglycosides is specific to MexXY-OprM, and extrusion of a group of the beta-lactams, i.e., carbenicillin, sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to MexAB-OprM . Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin, cefsulodin, and flomoxef, while MexXY-OprM does not . These substrate specificities are distinct from those reported previously. Antimicrob Agents Chemother, 2000 Dec, 44(12), 3317 - 21 Interactions of bacterial cationic peptide antibiotics with outer and cytoplasmic membranes of Pseudomonas aeruginosa; Zhang L et al.; Polymyxins B and E1 and gramicidin S are bacterium-derived cationic antimicrobial peptides . The polymyxins were more potent than gramicidin S against Pseudomonas aeruginosa, with MICs of 0.125 to 0 . 25 and 8 microg/ml, respectively . These peptides differed in their affinities for binding to lipopolysaccharide, but all were able to permeabilize the outer membrane of wild-type P . aeruginosa PAO1 strain H103, suggesting differences in their mechanisms of self-promoted uptake . Gramicidin S caused rapid depolarization of the bacterial cytoplasmic membrane at concentrations at which no killing was observed within 30 min, whereas, conversely, the concentrations of the polymyxins that resulted in rapid killing resulted in minimal depolarization . These data indicate that the depolarization of the cytoplasmic membrane by these peptides did not correlate with bacterial cell lethality. Acta Otorhinolaryngol Belg, 2000, 54(3), 367 - 72 Failure of local defense mechanisms in cystic fibrosis; Proesmans M et al.; Cystic fibrosis (CF) is an hereditary disease with pancreatic insufficiency and chronic respiratory tract infections leading to irreversible lung damage as its main features . The typical pathogen for the respiratory disease is Pseudomonas aeruginosa . Although the insight into the pathophysiology and genetics of CF have evolved over the past 10 years, there is not yet a full understanding of the increased susceptibility for respiratory infections, specifically with Pseudomonas aeruginosa . In this overview we elucidate the current knowledge and describe the existing hypothesis on the possible links between the pathophysiological defects and the failure of the local defense mechanism in CF airways. Plant Physiol, 2000 Nov, 124(3), 1159 - 68 Analysis of the alternative pathways for the beta-oxidation of unsaturated fatty acids using transgenic plants synthesizing polyhydroxyalkanoates in peroxisomes; Allenbach L et al.; Degradation of fatty acids having cis-double bonds on even-numbered carbons requires the presence of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle . Two alternative pathways have been described to degrade these fatty acids . One pathway involves the participation of the enzymes 2, 4-dienoyl-coenzyme A (CoA) reductase and Delta(3)-Delta(2)-enoyl-CoA isomerase, whereas the second involves the epimerization of R-3-hydroxyacyl-CoA via a 3-hydroxyacyl-CoA epimerase or the action of two stereo-specific enoyl-CoA hydratases . Although degradation of these fatty acids in bacteria and mammalian peroxisomes was shown to involve mainly the reductase-isomerase pathway, previous analysis of the relative activity of the enoyl-CoA hydratase II (also called R-3-hydroxyacyl-CoA hydro-lyase) and 2,4-dienoyl-CoA reductase in plants indicated that degradation occurred mainly through the epimerase pathway . We have examined the implication of both pathways in transgenic Arabidopsis expressing the polyhydroxyalkanoate synthase from Pseudomonas aeruginosa in peroxisomes and producing polyhydroxyalkanoate from the 3-hydroxyacyl-CoA intermediates of the beta-oxidation cycle . Analysis of the polyhydroxyalkanoate synthesized in plants grown in media containing cis-10-heptadecenoic or cis-10-pentadecenoic acids revealed a significant contribution of both the reductase-isomerase and epimerase pathways to the degradation of these fatty acids. Otolaryngol Head Neck Surg, 2000 Nov, 123(5), 617 - 23 Controlled multicenter study on chronic suppurative otitis media treated with topical applications of ciprofloxacin 0.2% solution in single-dose containers or combination of polymyxin B, neomycin, and hydrocortisone suspension; Miro N; Otic drops of either ciprofloxacin 0.2% solution (CIP) or a combination of polymyxin B, neomycin, and hydrocortisone suspension (PNH) were administered for 6 to 12 days to patients (14-71 years old) with chronic suppurative otitis media in a randomized, nonblinded, multicenter clinical trial . Two hundred thirty-two enrolled patients were analyzed for efficacy on a "per protocol" basis . The most frequently identified causal agents were Staphylococcus aureus (28% of the patients), Pseudomonas aeruginosa (19%), and Staphylococcus sp (9%) . Clinical success was observed in 91% and 87% of the CIP-and PNH-treated patients, respectively . At 1-month follow-up, 4% of CIP and 6% of PNH patients showed a relapse of otorrhea . Bacteriologic eradication was seen in 89% and 85% of patients in the CIP and PNH groups, respectively . At 1-month follow-up, reinfection or recurrence of infection appeared in 3 patients in the PNH group and in 1 patient in the CIP group . Both treatments were well tolerated . The most frequently reported adverse events were pruritus, stinging, and earache . Audiometric tests did not show changes attributable to study drugs in any but 1 patient in the PNH group . This clinical trial shows that topical 0.2% ciprofloxacin solution in single-dose containers is effective and well tolerated in patients with chronic suppurative otitis media. Am J Physiol Lung Cell Mol Physiol, 2000 Dec, 279(6), L1199 - 209 Keratinocyte growth factor protects against Pseudomonas aeruginosa-induced lung injury; Viget NB et al.; We have previously reported that keratinocyte growth factor (KGF) attenuates alpha-naphthylthiourea-induced lung injury by upregulating alveolar fluid transport . The objective of this study was to determine the effect of KGF pretreatment in Pseudomonas aeruginosa pneumonia . A 5% bovine albumin solution with 1 microCi of (125)I-labeled human albumin was instilled into the air spaces 4 or 24 h after intratracheal instillation of P . aeruginosa, and the concentration of unlabeled and labeled proteins in the distal air spaces over 1 h was used as an index of net alveolar fluid clearance . Alveolocapillary barrier permeability was evaluated with an intravascular injection of 1 microCi of (131)I-albumin . In early pneumonia, KGF increased lung liquid clearance (LLC) compared with that in nonpretreated animals . In late pneumonia, LLC was significantly reduced in the absence of KGF but increased above the control value with KGF . KGF pretreatment increased the number of polymorphonuclear cells recovered in the bronchoalveolar lavage fluid and decreased bacterial pulmonary translocation . In conclusion, KGF restores normal alveolar epithelial fluid transport during the acute phase of P . aeruginosa pneumonia and LLC in early and late pneumonia . Host response is also improved as shown by the increase in the alveolar cellular response and the decrease in pulmonary translocation of bacteria. Ter Arkh, 2000, 72(9), 54 - 7 {Some problems of the current therapy of infective endocarditis}; Butkevich OM et al.; AIM: To analyse clinical characteristics of endocarditis for the last 10 years, treatment difficulties and how to overcome them . MATERIAL AND METHODS: 135 patients with infectious endocarditis (IE) were examined according to the routine scheme using modern methods of diagnosis and therapy control: transthoracic and transesophageal echo-CG, test for antibiotics sensitivity of the microflora, etc . Immediate results were assessed in all the patients, some of them were followed up for maximum 5 years . RESULTS: Last decade was marked for growing difficulties in the treatment of IE related to its polyetiology . It can be caused by such therapy-resistant microbes as Staphylococcus aureus, Pseudomonas aeruginosa, anaerobic infection, nosocomial infection, injections of narcotic drugs, etc . CONCLUSION: Current course of IE dictates the necessity of fighting resistant microflora especially in case of nosocomial disease . Recurrences become more frequent . Indications to surgery did not change for the last decade . The best treatment results are achieved after antibacterial treatment of the valve. Rev Mal Respir, 2000 Aug, 17(3 Pt 2), 758 - 78 {Specific aspects and care of lung involvement in adults with cystic fibrosis}; Pin I et al.; Respiratory impairment is present in almost all adult cystic fibrosis patients and makes the prognosis . Viscous, infected and abundant secretions, inflammation and bronchial oedema, bronchoconstriction and respiratory muscle fatigue lead to airway obstruction, bronchiectasis and respiratory failure . The disease is preferentially located in the upper lobes . Exacerbations of the disease are due to bronchial infections and are often responsible for drops of the respiratory function . Regular spirometric surveillance is fundamental for the prognosis and the assessment of the effects of the treatment . Among adult patients chronic colonisation with mucoid and often multiresistant strains of Pseudomonas Aeruginosa are common . It is treated with i.v . high doses antibiotic courses and nebulized antibiotics between i.v . courses . Respiratory failure may require long term oxygen and non invasive mechanical ventilation . Systemic hypervascularization around the bronchiectasis may lead to moderate to severe hemoptysis, which may require embolization . Pneumothorax are associated with poor prognosis and are treated by pleural drainage and if failure by thoracoscopy. Rev Mal Respir, 2000 Aug, 17(3 Pt 2), 749 - 57 {Characteristics and specificities of cystic fibrosis in adults: evolutive disease of childhood or recently diagnosed disease?}; Hubert D et al.; We have studied the characteristics of 202 cystic fibrosis adult patients, all with chronic respiratory symptoms, with a median age of 27 yrs (18 to 55 yrs) and a male predominance (56%) . At genetic analysis, delta F508 homozygotes were 41%, delta F508 heterozygotes 42% and 17% had no delta F508 . The respiratory disease was more severe and complications were more frequent in adults: hemoptysis in 14%, pneumothorax in 15%, lung transplantation in 25 patients . Chronic bronchial colonisation with Pseudomonas aeruginosa, in 76% of patients, contributed to making treatments more severe because of antibiotic i.v . courses and nebulised antibiotics . Respiratory function showed a mean FVC of 62 +/- 22% and a mean FEVI of 48 +/- 94% . External pancreatic insufficiency was found in 83%, diabetes in 14% . Intestinal occlusion syndromes were observed in 11% of patients and hepatic cirrhosis in 8% . In spite of the severity of the respiratory disease, theses patients succeeded in social and occupational insertion; 62% were independent, 18% had children and 77% were working or studying . Analysis of the patients according to age at diagnosis showed that, in 38 patients diagnosed after the age of 18 yrs, the respiratory disease was less severe, pancreatic insufficiency and non-respiratory complications were less frequent (34% had pancreatic insufficiency, 5% had diabetes and none had cirrhosis) . This may partly be due to the presence of milder CFTR mutations . In conclusion, cystic fibrosis in adulthood frequently looks like an evolutive form of cystic fibrosis in childhood . Nevertheless, some late diagnosed forms in adults, with better prognosis, have been recently identified. Rev Mal Respir, 2000 Aug, 17(3 Pt 2), 725 - 32 {Diagnosis and management of cystic fibrosis in children}; Tamalet A et al.; Cystic fibrosis is a genetic disease occurring more frequently in Caucasians . The cystic fibrosis gene, cloned in 1999, codes for the cystic fibrosis transmembrane conductance regulator (CFTR) protein . Dysfunction of this protein leads to the clinical manifestations of cystic fibrosis, mainly lung disease and exocrine pancreas disorders . The pathophysiology of the respiratory component is quite complex, basically related to major and excessive inflammatory processes and to early microbial colonization . Respiratory physical therapy is a key element to management of the respiratory disorder . Antibiotic treatments should be adapted to the bacterial ecology, mainly using antistaphylococcal and antihaemophilus drugs initially, then directed against Pseudomonas aeruginosa . Other drugs including inhaled antiinflammatory drugs are currently in the evaluation stage . In addition, nutritional care and correction of pancreas insufficiency are necessary . The diagnosis of this disease must be made early although systematic neonatal screening is not proposed . Early diagnosis is necessary for improved care and prognosis . Currently, median survival is 29 years . This survival time should probably improve with better understanding of the pathophysiological mechanisms and new therapeutic perspectives. J Chromatogr B Biomed Sci Appl, 2000 Sep 15, 746(2), 161 - 72 Hydrophobic character of surface regions and total hydrophobicity of four variants of chromosomal class C beta-lactamase from Pseudomonas aeruginosa are identical . Chromatographic comparison of the hydrophobic character of the variants and the effect of focusing buffer composition on the separation of the variants by chromatofocusing with internal and external pH gradients; Walther-Rasmussen J et al.; The hydrophobic character of class C beta-lactamase molecular variants from Pseudomonas aeruginosa was compared by hydrophobic interaction chromatography and reversed-phase liquid chromatography, respectively . Separation of the variants by hydrophobic interaction chromatography was not achieved by modifying salt and pH of mobile phases . Reversed-phase liquid chromatography of the variants resulted in almost identical retention times . The results showed that the hydrophobic character of surface regions as well as total hydrophobicity of the variants are identical . The resolving power of external, internal and gradient chromatofocusing of the variants on strong and weak anion exchangers using low-molecular-mass buffers was compared to that of commercial ampholytes and showed no difference in separation pattern of the variants . Comparisons of variant isoelectric point (pI) values determined by chromatofocusing and isoelectric focusing showed that pI values determined by gradient chromatofocusing were most similar to the pI values determined by isoelectric focusing. J Bacteriol, 2000 Dec, 182(23), 6550 - 6 Role of Azotobacter vinelandii mucA and mucC gene products in alginate production; Nunez C et al.; Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts . In different bacterial species, the alternative sigma factor sigma(E) regulates the expression of functions related to the extracytoplasmic compartments . In A . vinelandii and Pseudomonas aeruginosa, the sigma(E) factor (AlgU) is essential for alginate production . In both bacteria, the activity of this sigma factor is regulated by the product of the mucA, mucB, mucC, and mucD genes . In this work, we studied the transcriptional regulation of the A . vinelandii algU-mucABCD gene cluster, as well as the role of the mucA and mucC gene products in alginate production . Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production. Clin Infect Dis, 2000 Nov, 31(5), 1131 - 3 Epub 2000 Nov 07. Cerebrospinal fluid penetration of high doses of intravenous ciprofloxacin in meningitis; Lipman J et al.; Nosocomial meningitis due to gram-negative organisms is a difficult clinical problem to manage because of both antibiotic resistance and poor penetration of many antimicrobials across the blood-brain barrier . Ciprofloxacin has potential in treating this condition when used in high doses . We investigated the plasma and cerebrospinal fluid (CSF) levels of ciprofloxacin in a patient with Pseudomonas aeruginosa meningitis who was treated with 400 mg of intravenous ciprofloxacin every 8 hours . Ciprofloxacin levels in plasma peaked at 10.29 mg/L without resulting in accumulation (8-hour trough levels, <1 mg/L), whereas the CSF level increased to 0.9 mg/L . This CSF level was confirmed to be similar 1 week later . After 1 week of therapy, during which there were no side effects attributable to ciprofloxacin, the organism was eradicated, and there was some clinical improvement . We recommend that 400 mg of intravenous ciprofloxacin every 8 hours be considered for treatment of difficult-to-treat gram-negative bacillary meningitis. Clin Infect Dis, 2000 Nov, 31(5), 1119 - 25 Epub 2000 Nov 06. Hospital outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-1, a novel transferable metallo-beta-lactamase; Cornaglia G et al.; A total of 8 Pseudomonas aeruginosa isolates was collected from 7 different patients in different wards of the University Hospital of Verona, Italy, from February 1997 to February 1998 . The high level of resistance to carbapenems (imipenem minimum inhibitory concentration was always >128 microg/mL) and other broad-spectrum beta-lactams and the rate of imipenem hydrolysis and its inhibition by ethylenediamine-tetra-acetic acid were all suggestive of production of a carbapenem-hydrolyzing metallo-beta-lactamase . A specific DNA probe derived from the recently cloned bla(VIM-1) gene hybridized to all the isolates . A genomic DNA fingerprinting profile revealed clonal relatedness for 7 of 8 isolates . A description of this hospital outbreak is reported, the occurrence of which confirms that proliferation of metallo-beta-lactamase-producing strains multiply resistant to beta-lactams is already a reality outside Japan . These findings emphasize the need for early recognition of similar isolates. J Hosp Infect, 2000 Nov, 46(3), 203 - 9 Assessment of in-vitro efficacy of 1% Virkon against bacteria, fungi, viruses and spores by means of AFNOR guidelines; Hernndez A et al.; Peroxygenic acid, under the brand name Virkon, has unleashed great debate following contradictory reports of its efficacy and spectrum of activity . The aim of this study was to test the biocidal activity of the compound against 10 different micro-organisms, following standard in-vitro test procedures . Bactericidal, fungicidal and sporicidal activities were determined using quantitative suspension and germ carrier tests and virucidal activity was assessed using a simple dilution suspension test, following the Association Francaise de Normalisation (AFNOR) guidelines . One percent Virkon demonstrated bactericidal activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus hirae and Mycobacterium smegmatis in the suspension test and against P . aeruginosa, E . coli, S . aureus and E . hirae in the carrier test . One percent Virkon showed virucidal activity against poliovirus in the suspension test . However, this concentration did not comply with sporicidal and fungicidal activity guidelines . In conclusion, 1% Virkon is effective only against vegetative bacteria, yeasts and viruses, and should therefore be considered a low-level disinfectant . J Leukoc Biol, 2000 Nov, 68(5), 700 - 6 Losartan, a selective inhibitor of subtype AT1 receptors for angiotensin II, inhibits neutrophil recruitment in the lung triggered by fMLP; Raiden S et al.; We have shown that losartan, a selective inhibitor of AT1 receptors for angiotensin II (AII), inhibits the binding of {3H}fMLP to neutrophil receptors (FPR) . Here, we analyze, in Wistar rats, the effect of losartan on neutrophil recruitment in the lung triggered by fMLP . We found that i.v . infusion of losartan (0.4-20.0 microg/kg/min) inhibits neutrophil recruitment induced by i.t . instillation of fMLP, without affecting the responses induced by other stimuli, such as aggregated human IgG (aIgG), precipitating immune complexes (IC), or zymosan . Histological evaluation of lungs as well as the analysis of lung hemorrhage indices showed that losartan prevents tissue injury partially in fMLP-challenged rats . We also analyzed the effect of losartan on lung-neutrophil recruitment triggered by i.t . instillation of Pseudomonas aeruginosa . Not only was there a marked decrease in neutrophil recruitment but also a significant increase in the survival of rats instillated with Pseudomonas aeruginosa, as a consequence of losartan treatment . Our results support the notion that losartan may be useful in the treatment of certain lung inflammatory disorders associated with bacterial infectious diseases. Respiration, 2000, 67(5), 477 - 90 Early detection of lung disease and its association with the nutritional status, genetic background and life events in patients with cystic fibrosis; Kraemer R et al.; Progression of lung disease is the most prominent cause of morbidity and death in patients with cystic fibrosis (CF), but the severity of lung disease and the rate of lung function decline are highly variable . An attempt was made to define accurate estimates of disease progression in these patients early diagnosed and prospectively evaluated until 10 years of age . The primary question to ask was whether functional abnormalities detected already in infancy are associated with functional derangements later on in life, and may be useful as parameters of prognostic value . Early diagnosis of CF can best be achieved by screening of mutation by new techniques (buccal cell brushing) in infants, even when the sweat test or accurate blood sampling is not available . Moreover, in infants lung function can be assessed by infant whole-body plethysmography enabling the study of the interrelationship with delayed weight gain and growth retardation, as well as the associations with the most common disease-causing mutations . Out of a cohort of 80 infants (39 males, 41 females) with CF a follow-up study was started with 50 CF infants diagnosed during infancy (mean age 4.6 +/- 4.0 months; range 0.1-12.7 months) and prospectively evaluated at 6-month intervals during the first 2 years of life . Moreover, in 32 CF children out of this cohort, follow-up was continued until 10 years of age . Differences were encountered with respect to the different events occurring during the first years of life, especially the onset of chronic colonization with Pseudomonas aeruginosa . The association between infant lung function and specific mutations (DeltaF508 homozygotes, frameshift DeltaF508/3905insT compound heterozygotes and nonsense DeltaF508/R553X compound heterozygotes) furthermore revealed that differences in lung function within the genetic groups are mainly related to the degree of pulmonary hyperinflation . Pulmonary hyperinflation was also associated with the degree of impaired nutritional status . An association between impaired gas exchange characteristics at 10 years of age and the degree of pulmonary hyperinflation during infancy finally demonstrates that by early mutation screening, lung function testing and assessment of the nutritional status predictors of disease progression later on in life can be defined . Therefore, preventive therapeutic measures should primarily be based on such prognostic factors . Mol Microbiol, 2000 Oct, 38(2), 213 - 31 The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage; Nakayama K et al.; Pseudomonas aeruginosa produces three types of bacteriocins: R-, F- and S-type pyocins . The S-type pyocin is a colicin-like protein, whereas the R-type pyocin resembles a contractile but non-flexible tail structure of bacteriophage, and the F-type a flexible but non-contractile one . As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage . In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P . aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages . The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with P2 phage and the F-type from lambda phage . This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages . The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins . A systematic polymerase chain reaction (PCR) analysis of P . aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity. Lett Appl Microbiol, 2000 Oct, 31(4), 313 - 8 Monoclonal antibody detection of naphthalene dioxygenase from Pseudomonas aeruginosa 2NR; Civilini M et al.; A monoclonal antibody, designated mAb alpha(CT), was generated against a peptide of the ISP(NAP) alpha-subunit of the naphthalene dioxygenase (NDO) enzyme of Pseudomonas aeruginosa . Since NDO expression is induced by aromatic hydrocarbons, its detection is important as a tool for environmental biomonitoring . This antibody is highly specific and works well both in an indirect ELISA assay and Western Blot analysis, allowing the detection of Pseudomonas spp . expressing the NDO inducible enzyme . The detection threshold for the ELISA assay developed in this work was 10(4) colony forming units (cfu) per ml . Thus, this mAb could represent a powerful tool to test for pollutants in soil, groundwater, and other natural environments. Microbiology, 2000 Nov, 146 ( Pt 11), 2803 - 14 Involvement of the rml locus in core oligosaccharide and O polysaccharide assembly in Pseudomonas aeruginosa; Rahim R et al.; L-Rhamnose (L-Rha) is a component of the lipopolysaccharide (LPS) core, several O antigen polysaccharides, and the cell surface surfactant rhamnolipid of Pseudomonas aeruginosa . In this study, four contiguous genes (rmlBDAC) responsible for the synthesis of dTDP-L-Rha in P . aeruginosa have been cloned and characterized . Non-polar chromosomal rmlC mutants were generated in P . aeruginosa strains PAO1 (serotype O5) and PAK (serotype O6) and LPS extracted from the mutants was analysed by SDS-PAGE and Western immunoblotting . rmlC mutants of both serotype O5 and serotype O6 synthesized a truncated core region which was unable to act as an attachment point for either A-band or B-band O antigen . A rmd rmlC PAO1 double mutant (deficient in biosynthesis of both D-Rha and L-Rha) was constructed to facilitate structural analysis of the mutant core region . This strain has an incomplete core oligosaccharide region and does not produce A-band O antigen . These results provide the genetic and structural evidence that L-Rha is the receptor on the P . aeruginosa LPS core for the attachment of O polysaccharides . This is the first report of a genetically defined mutation that affects the synthesis of a single sugar in the core oligosaccharide region of P . aeruginosa LPS, and provides further insight into the mechanisms of LPS biosynthesis and assembly in this bacterium. Pediatr Pulmonol, 2000 Nov, 30(5), 413 - 24 Mouse models of chronic lung infection with Pseudomonas aeruginosa: models for the study of cystic fibrosis; Stotland PK et al.; The discovery of the CFTR gene in 1989 has lead to rapid progress in understanding the molecular basis of cystic fibrosis (CF) and the biological properties of the cystic fibrosis transmembrane conductance regulator (CFTR) protein . However, more than 10 years later, recurrent lung infections with Pseudomonas aeruginosa, which lead to chronic lung disease and eventual respiratory failure, remain the major cause of morbidity and mortality among CF patients . A distinguishing feature of lung disease in CF is an exaggerated and persistent inflammatory response, characterized by the accumulation of excessive numbers of neutrophils and dysregulated cytokine production . The events leading to the establishment of lung infection with P . aeruginosa, especially the inflammatory and immunological events, and the relation between the CF defect and infection, remain largely undefined . Progress in this area has been hampered by the lack of a suitable animal model . An exciting achievement in the past few years has been the development of a number of variants of CFTR-deficient mice which exhibit defective cAMP-mediated Cl(-) conductance and have a range of clinical phenotypes from mild to severe . In parallel, a model of chronic P . aeruginosa lung infection has been established in genetically and immunologically well-defined inbred mouse strains which differ in susceptibility to this infection in the lung . BALB/c mice are resistant, while DBA/2 mice are extremely susceptible, with high mortality within 3 days of infection . C57BL/6 and A/J mice are relatively susceptible and experience low mortality . Furthermore, the bacterial load correlates with the magnitude and quality of the inflammatory response in the infected lungs of BALB/c and C57BL/6 mice . Although results of infection studies in CFTR-deficient mice have been variable, C57BL/6-Cftr(m1UNC)/Cftr(m1UNC) knockout mice compared to littermate control mice are highly susceptible to chronic P . aeruginosa infection in the lung . The availability of CFTR knockout mice and non-CF inbred mice differing in susceptibility to chronic P . aeruginosa infection offers useful tools for progress in understanding the genesis of chronic P . aeruginosa infection and the ensuing inflammation in the CF lung, as well as the relation between the CF defect and infection . Information generated from these studies will provide the rationale for the development of novel immunomodulatory measures capable of ameliorating or modulating the chronic inflammation associated with CF lung disease . FEMS Immunol Med Microbiol, 2000 Nov, 29(3), 227 - 32 The effect of pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa; Schultz MJ et al.; To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P . aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA . P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05) . P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody . The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection. FEMS Microbiol Lett, 2000 Nov 15, 192(2), 205 - 10 Respiratory pathways of Rhodobacter sphaeroides 2.4.1(T): identification and characterization of genes encoding quinol oxidases; Mouncey NJ et al.; Rhodobacter sphaeroides 2.4.1(T) respires aerobically via a branched respiratory chain consisting of both cytochrome c oxidases and quinol oxidases . Here, genes from chromosome II encoding two distinct quinol oxidases have been characterized . The qoxBA genes encode a putative heme-copper quinol oxidase, whereas the qxtAB genes encode a quinol oxidase homologous to the cyanide-insensitive oxidase of Pseudomonas aeruginosa . No phenotype was observed for mutations in either oxidase in the wild-type background . A strain containing a qxtA mutation in a cytochrome bc(1) complex mutant background was unable to grow aerobically . No role was found for the Qox oxidase, nor was a qoxB::lacZ transcriptional fusion expressed under a variety of conditions . These are the first molecular studies to characterize the quinol oxidases of R . sphaeroides 2.4.1(T). Harefuah, 2000 Oct, 139(7-8), 267 - 9, 327 {Corneal infection in wearers of contact lenses: causes, effect on visual acuity and prevention}; Domniz Y et al.; This is a 5-year retrospective survey of corneal infection in wearers of optical contact lenses (OCL) . 23 of the 61 patients (38%; Hasharon Hospital) with positive cultures wore OCL . Visual acuity improved in 15 (65%), no change was noted in 4 (17.5%) and there was deterioration in 4 (17.5%), as compare with status on admission . Pseudomonas aeruginosa was the most common cause of infections among OCL wearers . The improvement in visual acuity expected due to wearing OCL was affected by infections . Those after Staphylococcus albus infections had the highest rate (100%) of improvement in visual acuity and after Ps . aeruginosa the lowest rate (57.2%) of improvement, as well as the highest rate of deterioration (42.8%) found following recovery . OCL wearers are at higher risk for damage to visual acuity following corneal infection, and highly virulent infections in OCL wearers are responsible for a high risk of damage to visual acuity. J Antimicrob Chemother, 2000 Nov, 46(5), 733 - 9 A combined in vivo pharmacokinetic-in vitro pharmacodynamic approach to simulate target site pharmacodynamics of antibiotics in humans; Delacher S et al.; We describe a new approach to quantify in vivo anti-infective activity by simulating effect site pharmacokinetics of antibiotics in vitro . This approach is based on (i) the in vivo measurement of interstitial drug pharmacokinetics (PK) at the target site and (ii) a subsequent pharmacodynamic (PD) simulation of the time versus drug concentration profile in an in vitro setting . To demonstrate the feasibility of this approach, individual time-concentration profiles of ciprofloxacin were measured in the interstitial space fluid of eight healthy volunteers by microdialysis following iv administration of 200 mg . Thereafter, different isolates of Pseudomonas aeruginosa were exposed in vitro to the interstitial ciprofloxacin concentration profile obtained from in vivo experiments . This led to a 1- to 3-log10 decrease in the number of viable organisms after 8 h . Significant correlations were observed between the maximal bactericidal effect and several PK surrogate parameters, notably the AUC/MIC ratio (P: = 0.0005), the C:max/MIC ratio (P: = 0.006) and the time > MIC (P: = 0.02) . Furthermore, the data were analysed with an integrated PK-PD model allowing a much more detailed evaluation of the data than using MIC . The model employed an E:max relationship to link unbound ciprofloxacin concentration to bacterial kill rate . In conclusion, our experiments show that therapeutic success and failure in antimicrobial therapy may be explained by pharmacokinetic variability at the target site . Therefore, the in vivo PK-in vitro PD approach presented in our study may provide valuable guidance for drug and dose selection of antimicrobial agents. Pulm Pharmacol Ther, 2000, 13(6), 293 - 9 CGS 21680, dibutyryl cyclic AMP and rolipram attenuate the pro-inflammatory interactions of the Pseudomonas aeruginosa -derived pigment, 1-hydroxyphenazine, with human neutrophils; Ramafi G et al.; The effects of the intracellular adenosine 3':5' cyclic monophosphate (cAMP)-elevating agents, CGS 21680 (0.01- 1 microM) and rolipram (0.01-1 microM), as well as those of dibutyryl cAMP (0 . 05-4 mM) on the pro-inflammatory interactions of the P . aeruginosa -derived pigment, 1-hydroxyphenazine (1-hp, 3.1 and 12.5 microM), with human neutrophils have been investigated in vitro . Ca(2+)fluxes in FMLP-activated neutrophils were measured using a fura-2/AM spectrofluorimetric procedure, while a colourimetric method was used to measure release of the primary granule enzyme, elastase, from the cells . Treatment with 1-hp resulted in delayed clearance of Ca(2+)from the cytosol of N -formyl- L -methionyl- L -leucyl- L -phenylalanine (FMLP, 1 microM)-activated neutrophils and increased release of elastase . All 3 test agents caused dose-related antagonism of 1-hp-mediated potentiation of elastase release from activated neutrophils, which was associated with restoration of Ca(2+)homeostasis . These observations demonstrate the potential of cAMP-elevating agents, acting on Ca(2+)clearance mechanisms in activated neutrophils, to attenuate the potentially harmful pro-inflammatory effects of 1-hp . Schweiz Med Wochenschr, 2000 Sep 30, 130(39), 1366 - 72 {Inhaled colistin in cystic fibrosis}; Tamm M et al.; The clinical course of cystic fibrosis (CF) is characterised by chronic bronchial infection with Pseudomonas aeruginosa . Therapy with inhaled aminoglycosides was introduced to decrease the rate of infectious exacerbations and to delay pulmonary progression . However, development of resistance to aminoglycosides is frequent . Few investigations are available into the resistance profile under treatment with colistin . Antibiotic resistance to colistin was analysed in 44 adult CF patients treated with inhaled colistin . Resistance to aminoglycosides was observed in 86% of cases (38/44) before therapy and decreased to 43% (19/44) under treatment with colistin . Five patients (11%) developed polymyxin resistance . After cessation of therapy pseudomonas became sensitive to polymyxin within a few months and enabled colistin to be reintroduced . In addition, we performed a pilot study analysing the effect of inhaled colistin on the growth of pseudomonas . The number of Pseudomonas aeruginosa decreased from 16.7 million (CFU) bacteria per ml sputum to 2.9 million under therapy with colistin . There was a more than tenfold increase in bacterial counts after inhaled colistin was stopped . Genotyping revealed no change in the type of pseudomonas strains . CONCLUSION: Development of resistance to polymyxin is not rare under long-term treatment with inhaled colistin and requires temporary interruption of therapy . Sputum cultures should therefore be tested regularly for polymyxin resistance in patients treated with inhaled colistin. Am Fam Physician, 2000 Oct 15, 62(8), 1870 - 6 Topical fluoroquinolones for eye and ear; Morden NE et al.; Topical fluoroquinolones are now available for use in the eye and ear . Their broad spectrum of activity includes the common eye and ear pathogens Staphylococcus aureus and Pseudomonas aeruginosa . For the treatment of acute otitis externa, these agents are as effective as previously available otic preparations . For the treatment of otitis media with tympanic membrane perforation, topical fluoroquinolones are effective and safe . These preparations are approved for use in children, and lack of ototoxicity permits prolonged administration when necessary . Topical fluoroquinolones are not appropriate for the treatment of uncomplicated conjunctivitis where narrower spectrum agents suffice; they represent a simplified regimen for the treatment of bacterial keratitis (corneal ulcers) . When administered topically, fluoroquinolones are well tolerated and offer convenient dosing schedules . Currently, bacterial resistance appears limited. Crit Care, 2000, 4(4), 255 - 61 Epub 2000 Jul 07. Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid 'real-time' polymerase chain reaction; Pirnay JP et al.; STATEMENT OF FINDINGS: We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples . This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction . The time from sample collection to result was less than 1h . RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment. Appl Environ Microbiol, 2000 Nov, 66(11), 4735 - 41 Ingested blood contributes to the specificity of the symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech; Indergand S et al.; Hirudo medicinalis, the medicinal leech, usually carries in its digestive tract a pure culture of Aeromonas veronii bv . sobria . Such specificity is unusual for digestive tracts that are normally colonized by a complex microbial consortium . Important questions for the symbiotic interaction and for the medical application after microvascular surgery are whether other bacteria can proliferate or at least persist in the digestive tract of H . medicinalis and what factors contribute to the reported specificity . Using a colonization assay, we were able to compare experimentally the ability of clinical isolates and of a symbiotic strain to colonize H . medicinalis . The symbiotic A . veronii bv . sobria strain proliferated well and persisted for at least 7 days inside the digestive tract . In contrast, the proliferation of Pseudomonas aeruginosa and Staphylococcus aureus was inhibited inside the animal compared to growth in the in vitro control, indicating that the ingested blood was modified within the digestive tract . However, both strains were able to persist in the digestive tract for at least 7 days . For an Escherichia coli strain, the viable counts decreased approximately 1, 000-fold within 42 h . The decrease of viable E . coli could be prevented by interfering with the activation of the membrane-attack complex of the complement system that is present in blood . This suggests that the membrane-attack complex remained active inside H . medicinalis and prevented the proliferation of sensitive bacteria . Thus, antimicrobial properties of the ingested vertebrate blood contribute to the specificity of the A . veronii-H . medicinalis symbiosis, in addition to modifications of the blood inside the digestive tract of H . medicinalis. Pediatr Infect Dis J, 2000 Oct, 19(10), 959 - 63 Pseudomonas aeruginosa bacteremia in children: analysis of trends in prevalence, antibiotic resistance and prognostic factors; Grisaru-Soen G et al.; OBJECTIVE: To determine the factors predisposing to Pseudomonas aeruginosa bacteremia as well as the prevalence, source of infection, outcome and prognostic factors in pediatric patients . METHODS: Retrospective review of pediatric patients with P . aeruginosa bacteremia, at a large tertiary care hospital during a 6.5-year period . RESULTS: Seventy patients with P . aeruginosa bacteremia were identified . The annual rate of P . aeruginosa bacteremia remained unchanged during the study period . Antibiotic susceptibility remained unchanged except for two patients with extensive burns who developed resistant strains . Underlying diseases were malignancy (50%), prematurity (6%), burns (7%) and others (37%) . The overall mortality associated with P . aeruginosa bacteremia was 20% . The fatality rate was higher among the young infants (compared with older children) and those who received previous antibiotic therapy (P = 0.02) . Mortality rate was higher in nosocomial than in community-acquired infections (25% compared with 11.5%) . The mortality rate of low birth weight and burns patients was significantly higher when compared with oncology patients or other patients, 75 and 40% compared with 11 and 19%, P = 0.01 . Multiple regression analysis revealed a correlation only between the underlying disease and mortality (P = 0.02) . In the oncology patients the only significant risk factor for mortality was absolute neutrophil count < or =0.1 x 10(9)/l (P = 0.06) . CONCLUSION: P . aeruginosa bacteremia, although apparently not increasing in incidence and antibiotic resistance, is still a common serious complication in immunocompromised children with a high mortality rate . We conclude that the underlying disease is the main determinant of the clinical outcome. Am J Kidney Dis, 2000 Nov, 36(5), 1009 - 13 Analysis of microbiological trends in peritoneal dialysis-related peritonitis from 1991 to 1998; Zelenitsky S et al.; The microbial cause of peritoneal dialysis-related peritonitis is an important determinant of clinical outcome and the basis of widely used treatment guidelines . Five hundred forty-six cases of peritonitis in 374 patients from 1991 to 1998 were analyzed . The rate of peritonitis declined significantly from 1.37 episodes/patient-year in 1991 to 0.55 episode/patient-year in 1998 (P = 0.02) . The rate of Gram-positive peritonitis decreased significantly from 0.75 to 0.28 episode/patient-year during the same period (P = 0.02) . Conversely, the occurrence of Gram-negative peritonitis remained constant at approximately 0.16 episode/patient-year (P = 0.28) . Staphylococcus epidermidis and Staphylococcus aureus were the most common causes of peritonitis, isolated in 27.8% and 19.3% of the culture-positive cases, respectively . A distinct decrease in peritonitis caused by S epidermidis was observed, with 0.40 episode/patient-year in 1991 compared with 0.11 to 0.20 episode/patient-year during subsequent years . The rate of infections caused by S aureus decreased significantly over time from a high of 0.21 episode/patient-year in 1992 to a low of 0.04 episode/patient-year in 1998 (P = 0.01) . Pseudomonas aeruginosa, Escherichia coli, and KLEBSIELLA: species were the most common causes of Gram-negative peritonitis, identified in 7.1%, 6.8%, and 5.2% of culture-positive cases, respectively . The most dramatic increase in antibiotic resistance was seen among S epidermidis . From 1991 and 1992 to 1997 and 1998, resistance to ciprofloxacin increased from 5.4% to 47.8% (P = 0.003), and resistance to methicillin increased from 18.9% to 73.9% (P = 0.03) . Our study showed significant trends in the causative pathogens of peritoneal dialysis-related peritonitis and dramatic increases in antibiotic resistance . These data support further study and warrant reevaluation of current treatment practices. Chemotherapy, 2000 Nov-Dec, 46(6), 383 - 9 Comparison of the bactericidal activity of trovafloxacin and ciprofloxacin, alone and in combination with cefepime, against Pseudomonas aeruginosa; McNabb J et al.; BACKGROUND: Although ciprofloxacin exhibits more intense microbiological activity against Pseudomonas aeruginosa than does trovafloxacin, the clinical relevance of this observation remains questionable, particularly when the agents are combined with another antipseudomonal agent . METHODS: To evaluate this further, we conducted a four-way crossover trial to compare the bactericidal activities of ciprofloxacin and trovafloxacin, alone and in combination with cefepime, against three clinical isolates of P . aeruginosa . Healthy subjects received the following regimens, dosed to steady state: trovafloxacin 300 mg/24 h; ciprofloxacin 400 mg/12 h; trovafloxacin 300 mg/24 h plus cefepime 2 g/12 h, and ciprofloxacin 400 mg/12 h plus cefepime 2 g/12 h . Serum bactericidal titers were performed with each regimen . RESULTS: As monotherapy, the area under the bactericidal curve for ciprofloxacin exceeded that of trovafloxacin for all isolates . No significant difference in the overall degree of bactericidal activity was noted for two of three P . aeruginosa isolates for the combination regimens . Additionally, both combination regimens provided bactericidal activity for 100% of the dosing interval for all isolates . CONCLUSION: These results indicate that, while in vitro differences exist among these quinolones for P . aeruginosa, when a fluoroquinolone is combined with a beta-lactam, this is likely to be of little clinical significance . Acta Crystallogr D Biol Crystallogr, 2000 Nov, 56 ( Pt 11), 1501 - 4 The purification, crystallization and preliminary structural characterization of glucose-1-phosphate thymidylyltransferase (RmlA), the first enzyme of the dTDP-L-rhamnose synthesis pathway from Pseudomonas aeruginosa; Blankenfeldt W et al.; Glucose-1-phosphate thymidylyltransferase (RmlA; E.C . 2.7.7.24) is the first of four enzymes involved in the biosynthesis of dTDP-L-rhamnose, the precursor of L-rhamnose, a key component of the cell wall of many pathogenic bacteria . RmlA catalyses the condensation of thymidine triphosphate (dTTP) and alpha-D-glucose-1-phosphate (G1P), yielding dTDP-D-glucose . RmlA from Pseudomonas aeruginosa has been overexpressed and purified . Crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with PEG 6000 and lithium sulfate as precipitant . Several diffraction data sets of single frozen crystals were collected to a resolution of 1.66 A . Crystals belonged to space group P1, with unit-cell parameters a = 71.5, b = 73.1, c = 134.7 A, alpha = 89.9, beta = 80.9, gamma = 81.1 degrees . The asymmetric unit contains eight monomers in the form of two RmlA tetramers with a solvent content of 51% . Selenomethionine-labelled protein has been obtained and crystallized. Int J Antimicrob Agents, 2000 Oct, 16(2), 131 - 3 Empirical treatment of sepsis in neutropenic patients; Klastersky J; Febrile neutropenia remains a major cause of morbidity in cancer patients receiving chemotherapy . Although the mortality associated with febrile neutropenia has dramatically decreased over the last three decades, the overall death rate during and immediately after an episode of febrile neutropenia can be as high as 10% with half of the patients dying directly as a result of the infection itself . A series of developments has led to this marked reduction in mortality . Among them, a pivotal role has been played by the concept of hospital-based empirical therapy with broad-spectrum combinations of antibiotics, aimed primarily against Gram-negative organisms, namely Pseudomonas aeruginosa Int J Antimicrob Agents, 2000 Oct, 16(2), 103 - 6 Therapeutic guidelines for Pseudomonas aeruginosa infections; Giamarellou H; Pseudomonas aeruginosa nowadays is encountered among the leading pathogen in (i) ICU pneumonia; (ii) nosocomial bacteremia and AIDS primary bacteremia; (iii) iv drug users endocarditis; (iv) exacerbations of cystis fibrosis; (v) malignant external otitis and 'swimmers's ear', and (vi) contact lenses keratitis and traumatic endophthalmitis . The most vulnerable nosocomial hosts are the neutropenics and the mechanically ventilated patients in whom mortality rate exceeds 30% . Virulence of P . aeruginosa is attributed to the elaboration of various enzymes and toxins . There is also worldwide emergence of multiresistant phenotypes to antipseudomonal antibiotics . Therapeutic guidelines should therefore be based on (i) continuous resistance surveillance; (ii) in vitro synergistic interactions of antibacterial agents; (iii) pharmacodynamic properties of antibiotics interpreted by optimal dosing and appropriate frequency of administration; and (iv) current information on the necessity for combination therapy using an aminoglycoside. J Bacteriol, 2000 Nov, 182(22), 6401 - 11 The Pseudomonas aeruginosa lectins PA-IL and PA-IIL are controlled by quorum sensing and by RpoS; Winzer K et al.; In Pseudomonas aeruginosa, many exoproduct virulence determinants are regulated via a hierarchical quorum-sensing cascade involving the transcriptional regulators LasR and RhlR and their cognate activators, N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL) . In this paper, we demonstrate that the cytotoxic lectins PA-IL and PA-IIL are regulated via quorum sensing . Using immunoblot analysis, the production of both lectins was found to be directly dependent on the rhl locus while, in a lasR mutant, the onset of lectin synthesis was delayed but not abolished . The PA-IL structural gene, lecA, was cloned and sequenced . Transcript analysis indicated a monocistronic organization with a transcriptional start site 70 bp upstream of the lecA translational start codon . A lux box-type element together with RpoS (sigma(S)) consensus sequences was identified upstream of the putative promoter region . In Escherichia coli, expression of a lecA::lux reporter fusion was activated by RhlR/C4-HSL, but not by LasR/3O-C12-HSL, confirming direct regulation by RhlR/C4-HSL . Similarly, in P . aeruginosa PAO1, the expression of a chromosomal lecA::lux fusion was enhanced but not advanced by the addition of exogenous C4-HSL but not 3O-C12-HSL . Furthermore, mutation of rpoS abolished lectin synthesis in P . aeruginosa, demonstrating that both RpoS and RhlR/C4-HSL are required . Although the C4-HSL-dependent expression of the lecA::lux reporter in E . coli could be inhibited by the presence of 3O-C12-HSL, this did not occur in P . aeruginosa . This suggests that, in the homologous genetic background, 3O-C12-HSL does not function as a posttranslational regulator of the RhlR/C4-HSL-dependent activation of lecA expression. Cytokine, 2000 Nov, 12(11), 1662 - 8 Role of cytokine-induced neutrophil chemoattractant-2 (CINC-2) alpha in a rat model of chronic bronchopulmonary infections with Pseudomonas aeruginosa; Amano H et al.; In order to investigate the role of the cytokine-induced neutrophil chemoattractant (CINC) in chronic bronchopulmonary infection, we developed a rat model of bronchopulmonary infection with Pseudomonas aeruginosa by using the agar bead method, and determined the kinetics of bacterial and cell number, as well as the concentrations of CINC-1, CINC-2, and CINC-3 in bronchoalveolar lavage (BAL) fluids in this model . The bacterial number in the lung rapidly increased from days 1 to 4, and declined 14 days after challenge . Neutrophil number in BAL fluid increased up to one day after challenge, and then slowly decreased during 14 days post-challenge . Among the CINCs, the local production of CINC-2 alpha sharply increased at day 1 and then decreased until day 4 post-challenge, while the local production of CINC-1 slightly increased at day 1 post-challenge . Neither CINC-2 beta nor CINC-3 were detected during the entire course of the infection . Increased CINC-2 mRNA expression in the lung tissue after challenge was associated with CINC-2 alpha production in BAL fluid . Moreover, an immunohistochemical study demonstrated the localization of CINC-1 and CINC-2 alpha primarily in alveolar macrophages and, to a much lesser extent, in bronchial epithelium of infected lung tissues, whereas CINC-2 beta and CINC-3 were not detected . When anti-CINC-1 or anti-CINC-2 alpha polyclonal antibodies were used for neutralizing neutrophil chemotactic activities in BAL fluids, the anti-CINC-2 alpha antibody inhibited 70% of the chemotactic activity in BAL fluids from infected rats at day 1 after challenge . No inhibition was observed by anti-CINC-1 antibody . These data indicate that CINC-2 alpha, which is produced by alveolar macrophages and bronchial epithelial cells, plays a pivotal role in neutrophil accumulation in the airway of a rat model of chronic bronchopulmonary infection with P . aeruginosa . Braz J Med Biol Res, 2000 Nov, 33(11), 1275 - 82 Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity; Matsui T et al.; The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes . Expression of mycobacterial PLC-a and PLC-b in E . coli and M . smegmatis has been reported, whereas expression of the native proteins in M . tuberculosis H37Rv has not been demonstrated . The objective of the present study was to demonstrate that native PLC-a is expressed in M . tuberculosis H37Rv . Sera from mice immunized with recombinant PLC-a expressed in E . coli were used in immunoblots to evaluate PLC-a expression . The immune serum recognized a 49-kDa protein in immunoblots against M . tuberculosis extracts . No bands were visible in M . tuberculosis culture supernatants or extracts from M . avium, M . bovis and M . smegmatis . A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of beta-galactosidase activity . beta-Galactosidase activity was detected in M . smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages . The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive . In conclusion, expression of non-secreted native PLC-a was demonstrated in M . tuberculosis. Clin Infect Dis, 2000 Oct, 31(4), E9 - E14 Epub 2000 Oct 25. Large outbreak in a surgical intensive care unit of colonization or infection with Pseudomonas aeruginosa that overexpressed an active efflux pump; Bertrand X et