pyoverdin and Cystic-Fibrosis

Topics: Cystic Fibrosis; Iron; Oligopeptides; Pigments, Biological; Pseudomonas aeruginosa

The repurposing of gallium nitrate as an antibacterial, a drug used previously for the treatment of hypercalcemia, is a plausible alternative to combat infections by Pseudomonas aeruginosa, since it has antipseudomonal properties in vitro and in vivo in animal models and in human lung infections. Furthermore, gallium nitrate tolerance in clinical isolates is very rare. Nevertheless, studies on the reference strains PA14 and PAO1 show that resistance against gallium nitrate is achieved by decreasing gallium intracellular levels by increasing the production of pyocyanin. In this work, we induced resistance in a cystic fibrosis P. aeruginosa isolate and explored its resistance mechanisms. This isolated strain, INP-58M, was not a pyocyanin producer, and its pyoverdine levels remained unchanged upon gallium addition. However, it showed higher activities of NADPH-producing enzymes and the antioxidant enzyme SOD when gallium was added, which suggests a better antioxidant response. Remarkably, gallium intracellular levels in the resistant isolate were higher than those of the parental strain at 20 h but lower after 24 h of culture, suggesting that this strain is capable of gallium efflux.

Topics: Anti-Bacterial Agents; Cystic Fibrosis; Drug Repositioning; Drug Resistance, Bacterial; Gallium; Humans; Oligopeptides; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine

Topics: Antibiosis; Aspergillus fumigatus; Bacterial Proteins; Biofilms; Cystic Fibrosis; Humans; Iron; Mutation; Oligopeptides; Peptide Synthases; Pseudomonas aeruginosa; Trans-Activators; Virulence

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

Topics: Alginates; Animals; Anti-Bacterial Agents; Azithromycin; Bacterial Proteins; Berberine; Biofilms; Chitinases; Cyclophosphamide; Cystic Fibrosis; DNA, Bacterial; Drug Combinations; Drug Synergism; Glucuronic Acid; Hexuronic Acids; Humans; Lung; Metalloendopeptidases; Metalloproteases; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Neutropenia; Oligopeptides; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Virulence Factors

Pseudomonas aeruginosa typically displays loss of virulence-associated secretions over the course of chronic cystic fibrosis infections. This has led to the suggestion that virulence is a costly attribute in chronic infections. However, previous reports suggest that overproducing (OP) virulent pathotypes can coexist with non-producing mutants in the CF lung for many years. The consequences of such within-patient phenotypic diversity for the success of this pathogen are not fully understood. Here, we provide in-depth quantification of within-host variation in the production of three virulence associated secretions in the Liverpool cystic fibrosis epidemic strain of P. aeruginosa, and investgate the effect of this phenotypic variation on virulence in acute infections of an insect host model.. Within-patient variation was present for all three secretions (pyoverdine, pyocyanin and LasA protease). In two out of three patients sampled, OP isolates coexisted with under-producing mutants. In the third patient, all 39 isolates were under-producers of all three secretions relative to the transmissible ancestor LESB58. Finally, this phenotypic variation translated into variation in virulence in an insect host model.. Within population variation in the production of P. aeruginosa virulence-associated secretions can lead to high virulence sub-populations persisting in patients with chronic CF infections.

Topics: Adult; Animals; Bacterial Proteins; Chronic Disease; Cystic Fibrosis; Disease Models, Animal; Female; Humans; Insecta; Lung; Metalloproteases; Mutation; Oligopeptides; Phenotype; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Virulence; Virulence Factors

Laboratory experiments show that social interactions between bacterial cells can drive evolutionary change at the population level, but significant challenges limit attempts to assess the relevance of these findings to natural populations, where selection pressures are unknown. We have increasingly sophisticated methods for monitoring phenotypic and genotypic dynamics in bacteria causing infectious disease, but in contrast, we lack evidence-based adaptive explanations for those changes. Evolutionary change during infection is often interpreted as host adaptation, but this assumption neglects to consider social dynamics shown to drive evolutionary change in vitro. We provide evidence to show that long-term behavioral dynamics observed in a pathogen are driven by selection to outcompete neighboring conspecific cells through social interactions. We find that Pseudomonas aeruginosa bacteria, causing lung infections in patients with cystic fibrosis, lose cooperative iron acquisition by siderophore production during infection. This loss could be caused by changes in iron availability in the lung, but surprisingly, we find that cells retain the ability to take up siderophores produced by conspecifics, even after they have lost the ability to synthesize siderophores. Only when cooperative producers are lost from the population is the receptor for uptake lost. This finding highlights the potential pitfalls of interpreting loss of function in pathogenic bacterial populations as evidence for trait redundancy in the host environment. More generally, we provide an example of how sequence analysis can be used to generate testable hypotheses about selection driving long-term phenotypic changes of pathogenic bacteria in situ.

Topics: Adaptation, Physiological; Adolescent; Adult; Child; Child, Preschool; Cystic Fibrosis; Databases, Genetic; Denmark; Disease Susceptibility; Female; Genes, Bacterial; Humans; Infant; Iron; Lung; Male; Microbial Interactions; Molecular Sequence Data; Oligopeptides; Pseudomonas aeruginosa; Sequence Alignment; Virulence; Young Adult

Pseudomonas aeruginosa infection is problematic in patients with cystic fibrosis (CF). P. aeruginosa secretes a diversity of pigments, such as pyocyanin and pyoverdine. The aim of this study was to evaluate the effects of complexes of nickel(II) ([Ni(iaa)2(H2O)2]·H2O (iaa = imidazole-4-acetate anion), [Ni(1-allim)6](NO3)2 (1-allim = 1-allylimidazole) and NiCl2 on pyocyanin and pyoverdine production by 23 strains of P. aeruginosa isolated from cystic fibrosis under growth conditions specific for the CF respiratory system. The antibacterial effects and biophysical properties of the tested substances were measured by spectrofluorometric techniques, as well as by laser interferometry, confocal and atomic force microscopy. The cytotoxic properties of all compounds were measured by Annexin/IP assay against A549 cells. All tested compounds have no effect on pyocyanin production and decrease the pyoverdine secretion in about 40% of tested P. aeruginosa strains at non-cytotoxic range of concentrations. Imidazole-4-acetate anion and 1-allylimidazole have good diffusion properties in the mature P. aeruginosa PAO1 biofilm. In conclusion, the tested nickel(II) complexes do not have clinical implications in P. aeruginosa eradication in cystic fibrosis. The diffusion properties of 1-allylimidazole and imidazole-4-acetate and their lack of effect on A549 cells suggest that they might be considered for chemical synthesis with other transition metals.

Topics: Cell Line; Cystic Fibrosis; Humans; Imidazoles; Microscopy, Atomic Force; Nickel; Oligopeptides; Pseudomonas aeruginosa; Pyocyanine; Spectrometry, Fluorescence

Pseudomonas aeruginosa, is an opportunistic, bacterial pathogen causing persistent and frequently fatal infections of the lung in patients with cystic fibrosis. Isolates from chronic infections differ from laboratory and environmental strains in a range of traits and this is widely interpreted as the result of adaptation to the lung environment. Typically, chronic strains carry mutations in global regulation factors that could effect reduced expression of social traits, raising the possibility that competitive dynamics between cooperative and selfish, cheating strains could also drive changes in P. aeruginosa infections. We compared the expression of cooperative traits - biofilm formation, secretion of exo-products and quorum sensing (QS) - in P. aeruginosa isolates that were estimated to have spent different lengths of time in the lung based on clinical information. All three exo-products involved in nutrient acquisition were produced in significantly smaller quantities with increased duration of infection, and patterns across four QS signal molecules were consistent with accumulation over time of mutations in lasR, which are known to disrupt the ability of cells to respond to QS signal. Pyocyanin production, and the proportion of cells in biofilm relative to motile, free-living cells in liquid culture, did not change. Overall, our results confirm that the loss of social behaviour is a consistent trend with time spent in the lung and suggest that social dynamics are potentially relevant to understanding the behaviour of P. aeruginosa in lung infections.

Topics: Biofilms; Cell Communication; Cystic Fibrosis; Extracellular Space; Humans; Lung; Oligopeptides; Pancreatic Elastase; Peptide Hydrolases; Pseudomonas aeruginosa; Siderophores

Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections, most notably by Pseudomonas aeruginosa, which persists for decades in the lungs and undergoes extensive evolution. P. aeruginosa requires iron for virulence and uses the fluorescent siderophore pyoverdine to scavenge and solubilize ferric iron during acute infections. Pyoverdine mutants accumulate in the lungs of some CF patients, however, suggesting that the heme and ferrous iron acquisition pathways of P. aeruginosa are more important in this environment. Here, we sought to determine how evolution of P. aeruginosa in the CF lung affects iron acquisition and regulatory pathways through the use of longitudinal CF isolates. These analyses demonstrated a significant reduction of siderophore production during the course of CF lung infection in nearly all strains tested. Mass spectrometry analysis of one of these strains showed that the later CF isolate has streamlined the metabolic flux of extracellular heme through the HemO heme oxygenase, resulting in more-efficient heme utilization. Moreover, gene expression analysis shows that iron regulation via the PrrF small RNAs (sRNAs) is enhanced in the later CF isolate. Finally, analysis of P. aeruginosa gene expression in the lungs of various CF patients demonstrates that both PrrF and HemO are consistently expressed in the CF lung environment. Combined, these results suggest that heme is a critical source of iron during prolonged infection of the CF lung and that changes in iron and heme regulatory pathways play a crucial role in adaptation of P. aeruginosa to this ever-changing host environment.

Topics: Adaptation, Physiological; Adolescent; Bacterial Proteins; Child; Child, Preschool; Cystic Fibrosis; Gene Expression Regulation, Bacterial; Homeostasis; Humans; Iron; Mutation; Oligopeptides; Pigments, Biological; Pseudomonas aeruginosa; Pseudomonas Infections; Young Adult

Pseudomonas aeruginosa establishes life-long chronic infections in the cystic fibrosis (CF) lung by utilizing various adaptation strategies. Some of these strategies include altering metabolic pathways to utilize readily available nutrients present in the host environment. The airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphatidylcholine, a major component of lung surfactant. Pseudomonas aeruginosa can degrade phosphatidylcholine to glycerol and fatty acids to increase the availability of usable carbon sources in the CF lung. In this study, we show that some CF-adapted P. aeruginosa isolates utilize glycerol more efficiently as a carbon source than nonadapted isolates. Furthermore, a mutation in a gene required for glycerol utilization impacts the production of several virulence factors in both acute and chronic isolates of P. aeruginosa. Taken together, the results suggest that interference with this metabolic pathway may have potential therapeutic benefits.

Topics: Adaptation, Physiological; Culture Media; Cystic Fibrosis; Genes, Bacterial; Glycerol; Glycerolphosphate Dehydrogenase; Humans; Mutation; Oligopeptides; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Virulence Factors

The lungs of patients with cystic fibrosis become chronically infected with the bacterium Pseudomonas aeruginosa, which heralds progressive lung damage and a decline in health. Iron is a crucial micronutrient for bacteria and its acquisition is a key factor in infection. P. aeruginosa can acquire this element by secreting pyoverdine and pyochelin, iron-chelating compounds (siderophores) that scavenge iron and deliver it to the bacteria. Siderophore-mediated iron uptake is generally considered a key factor in the ability of P. aeruginosa to cause infection. We have investigated the amounts of pyoverdine in 148 sputum samples from 36 cystic fibrosis patients (30 infected with P. aeruginosa and 6 as negative controls). Pyoverdine was present in 93 samples in concentrations between 0.30 and 51 μM (median 4.6 μM) and there was a strong association between the amount of pyoverdine and the number of P. aeruginosa present. However, pyoverdine was not present, or below the limits of detection (~0.3 μM), in 21 sputum samples that contained P. aeruginosa. Pyochelin was also absent, or below the limits of detection (~1 μM), in samples from P. aeruginosa-infected patients with little or no detectable pyoverdine. Our data show that pyoverdine is an important iron-scavenging molecule for P. aeruginosa in many cystic fibrosis patients, but other P. aeruginosa iron-uptake systems must be active in some patients to satisfy the bacterial need for iron.

Topics: Adult; Cystic Fibrosis; Female; Humans; Iron; Male; Oligopeptides; Phenols; Pseudomonas aeruginosa; Pseudomonas Infections; Siderophores; Sputum; Thiazoles; Young Adult

This study was carried out to examine the antimicrobial activity of the aqueous extract of Panax quinquefolius from North American ginseng (NAGE) root against Pseudomonas aeruginosa . The minimum inhibitory concentrations of reference and clinical isolates of Pseudomonas aeruginosa were measured by a standard agar-dilution method. At subinhibitory NAGE concentrations, the secretion of virulence factors, motility on agar, and adhesion to 96-well microplates were studied on the nonmucoid Pseudomonas aeruginosa O1 strain. At suprainhibitory concentrations, the activity of NAGE against mature biofilm complexes formed in the Calgary Biofilm Device and the Stovall flow cell were assessed. NAGE possessed an antibacterial activity against all the Pseudomonas aeruginosa strains at 1.25%-2.5% w/v. NAGE also significantly attenuated pyocyanin, pyoverdine, and lipase concentrations, stimulated twitching, and attenuated swarming and swimming motility. At 1.25% w/v, NAGE augmented adhesion, and at 5% w/v detached 1-day-old biofilms in microplates. The extract also eradicated 6-day-old mature biofilms (5% w/v), and fluorescence microscopy displayed a reduction of live cells and biofilm complexes compared with nontreated biofilms. These data suggest that the aqueous extract from North American ginseng possesses antimicrobial activities in vitro.

Topics: Anti-Bacterial Agents; Biofilms; Cystic Fibrosis; Humans; Microbial Sensitivity Tests; Oligopeptides; Panax; Phytotherapy; Plant Extracts; Plant Roots; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Virulence; Virulence Factors

Whole-genome shotgun sequencing was used to study the sequence variation of three Pseudomonas aeruginosa isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1. The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent. Conversely, approximately 10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone. While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity. We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing. A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype. Overall, we conclude that most of the PAO1 genome represents a core P. aeruginosa backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes. Approximately half of these additional sequences are novel.

Topics: Adolescent; Bacterial Proteins; Child, Preschool; Cystic Fibrosis; Genetic Variation; Genome, Bacterial; Humans; Molecular Sequence Data; O Antigens; Oligopeptides; Pigments, Biological; Pseudomonas aeruginosa; Pseudomonas Infections; Sequence Analysis, DNA; Water Microbiology

Pseudomonas aeruginosa produces, under conditions of iron limitation, a high-affinity siderophore, pyoverdine (PVD), which is recognized at the level of the outer membrane by a specific TonB-dependent receptor, FpvA. So far, for P. aeruginosa, three different PVDs, differing in their peptide chain, have been described (types I-III), but only the FpvA receptor for type I is known. Two PVD-producing P. aeruginosa strains, one type II and one type III, were mutagenized by a mini-TnphoA3 transposon. In each case, one mutant unable to grow in the presence of the strong iron chelator ethylenediaminedihydroxyphenylacetic acid (EDDHA) and the cognate PVD was selected. The first mutant, which had an insertion in the pvdE gene, upstream of fpvA, was unable to take up type II PVD and showed resistance to pyocin S3, which is known to use type II FpvA as receptor. The second mutant was unable to take up type III PVD and had the transposon insertion in fpvA. Cosmid libraries of the respective type II and type III PVD wild-type strains were constructed and screened for clones restoring the capacity to grow in the presence of PVD. From the respective complementing genomic fragments, type II and type III fpvA sequences were determined. When in trans, type II and type III fpvA restored PVD production, uptake, growth in the presence of EDDHA and, in the case of type II fpvA, pyocin S3 sensitivity. Complementation of fpvA mutants obtained by allelic exchange was achieved by the presence of cognate fpvA in trans. All three receptors posses an N-terminal extension of about 70 amino acids, similar to FecA of Escherichia coli, but only FpvAI has a TAT export sequence at its N-terminal end.

Topics: Amino Acid Sequence; Bacterial Outer Membrane Proteins; Culture Media; Cystic Fibrosis; Ethylenediamines; Genetic Complementation Test; Humans; Iron; Molecular Sequence Data; Mutation; Oligopeptides; Pigments, Biological; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocins; Receptors, Cell Surface; Sequence Analysis, DNA

Pseudomonas aeruginosa strains from the chronic lung infections of cystic fibrosis (CF) patients are phenotypically and genotypically diverse. Using strain PAO1 whole genome DNA microarrays, we assessed the genomic variation in P. aeruginosa strains isolated from young children with CF (6 months to 8 years of age) as well as from the environment. Eighty-nine to 97% of the PAO1 open reading frames were detected in 20 strains by microarray analysis, while subsets of 38 gene islands were absent or divergent. No specific pattern of genome mosaicism defined strains associated with CF. Many mosaic regions were distinguished by their low G + C content; their inclusion of phage related or pyocin genes; or by their linkage to a vgr gene or a tRNA gene. Microarray and phenotypic analysis of sequential isolates from individual patients revealed two deletions of greater than 100 kbp formed during evolution in the lung. The gene loss in these sequential isolates raises the possibility that acquisition of pyomelanin production and loss of pyoverdin uptake each may be of adaptive significance. Further characterization of P. aeruginosa diversity within the airways of individual CF patients may reveal common adaptations, perhaps mediated by gene loss, that suggest new opportunities for therapy.

Topics: Adaptation, Physiological; Base Composition; Child; Child, Preschool; Cystic Fibrosis; DNA, Bacterial; Gene Transfer, Horizontal; Genetic Variation; Genome, Bacterial; Humans; Infant; Melanins; Oligonucleotide Array Sequence Analysis; Oligopeptides; Pigments, Biological; Pseudomonas aeruginosa; Pseudomonas Infections; Pseudomonas Phages; Pyocins; RNA, Transfer; Sequence Deletion

The lungs of cystic fibrosis patients are frequently colonized by Pseudomonas aeruginosa, which produces high-affinity fluorescent peptidic siderophores, pyoverdines. Three pyoverdines which differ in their peptide chain and are easily differentiated by isoelectric focusing exist, only one being produced by a given strain. P. aeruginosa isolates from cystic fibrosis patients of a German hospital were analyzed by sequential, pulse-field gel electrophoresis (PFGE) and for pyoverdine production and type. Only producers of type I and type II pyoverdine were found. There was a perfect correlation between the type of pyoverdine produced and the clonality determined by PFGE. PFGE clone C, the most prevalent among cystic fibrosis patients, and found in an aquatic environment, produced type II pyoverdine. Pyoverdine-negative mutants seemed to increase as a function of the lung colonization time, but retained the capacity to take up pyoverdines. Most isolates that took up type II pyoverdine were also able to utilize type I pyoverdine as judged by growth stimulation experiments. No correlation was observed between the loss of pyoverdine production and mucoidy.

Topics: Cystic Fibrosis; Electrophoresis, Gel, Pulsed-Field; Humans; Isoelectric Focusing; Mutation; Oligopeptides; Pharynx; Pigments, Biological; Pseudomonas aeruginosa; Pseudomonas Infections; Siderophores

Pyocin S3 was found to kill exclusively Pseudomonas aeruginosa isolates producing type II pyoverdine (exemplified by strain ATCC 27853). Killing was specifically inhibited by addition of type II ferripyoverdine. All Tn5 mutants resistant to pyocin S3 were defective for pyoverdine-mediated iron uptake and failed to produce an 85-kDa iron-repressed outer membrane protein. We conclude that this protein is probably the type II ferripyoverdine receptor that is used by pyocin S3 to gain entry into the cell.

Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Biological Transport; Cell Membrane; Cystic Fibrosis; Humans; Iron; Iron Chelating Agents; Mutagenesis, Insertional; Oligopeptides; Pigments, Biological; Pseudomonas aeruginosa; Pyocins

Sputum samples from the lungs of cystic fibrosis patients harboring Pseudomonas aeruginosa infections were collected and examined for the presence of the siderophore pyoverdine. Fluorescence quenching, due to the addition of ferric ion, as well as column and thin-layer chromatography results indicated that all samples contained the siderophore. Six samples furnished sufficient material after purification to allow us to obtain visible absorbance spectra. These spectra were characteristic of the ferrated analog of the P. aeruginosa pyoverdine, that is, ferripyoverdine, and in all cases they indicated a degree of ferration in excess of 50%. P. aeruginosa in the cystic fibrosis lung is thus iron stressed and responds by synthesizing pyoverdine, which subsequently binds ferric ion.

Topics: Adolescent; Adult; Child; Cystic Fibrosis; Female; Humans; Iron; Iron Chelating Agents; Male; Oligopeptides; Pigments, Biological; Pseudomonas Infections; Siderophores; Spectrum Analysis; Sputum

Nonmucoid Pseudomonas aeruginosa responds to iron deprivation by synthesizing the siderophores pyochelin and pyoverdine. When grown in iron-deficient medium, six mucoid P. aeruginosa strains isolated from cystic fibrosis patients synthesized copious amounts of the exopolysaccharide alginate. A procedure that eliminated the interference of alginate was developed so that siderophores could be extracted from the growth medium. All six isolates were then noted to produce both pyoverdine and pyochelin. This report thus confirms that mucoid P. aeruginosa, like its nonmucoid counterparts, elicits the siderophores commonly cited as those of the microbe.

Topics: Cystic Fibrosis; Iron Chelating Agents; Oligopeptides; Phenols; Pigments, Biological; Pseudomonas aeruginosa; Pseudomonas Infections; Siderophores; Thiazoles

The effect of Pseudomonas aeruginosa alkaline protease (AP), elastase (Ela), and the elastase from polymorphonuclear leukocytes (PMN Ela) on iron acquisition of pyoverdin from human transferrin and lactoferrin at physiologic pH was investigated. Incubation of iron-loaded transferrin with iron-free pyoverdin for 10 h at 40 degrees C in the presence of Ela yielded pyoverdin-iron(III) complex, in contrast to incubations of transferrin with pyoverdin alone, AP, or PMN Ela. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the incubations revealed fragmentation of transferrin by Ela in peptides smaller than 14,000 daltons, whereas AP and PMN Ela cleaved transferrin in fragments of 49,000 d and 43,000 d, respectively. Incubations of lactoferrin with the proteases and pyoverdin or pyoverdin alone did not result in iron acquisition by pyoverdin; however, lactoferrin was fragmented by Ela and AP.

Topics: Bacterial Proteins; Cystic Fibrosis; Endopeptidases; Humans; In Vitro Techniques; Iron Chelating Agents; Lactoferrin; Lactoglobulins; Metalloendopeptidases; Neutrophils; Oligopeptides; Pigments, Biological; Pseudomonas aeruginosa; Serine Endopeptidases; Transferrin

A cost-effective and more rapid means of detection of Pseudomonas aeruginosa in cultures from clinical specimens would be very advantageous. We have developed a modified MacConkey agar (MMA), which enhances pigment production of P. aeruginosa and which, if pyocyanin pigment is present, provides a relatively rapid and very cost-effective identification. The MMA medium inhibits the gram-positive organisms, while lactose- and non-lactose-fermenting gram-negative rods are easily distinguishable from pigment-producing pseudomonads. Organisms that produce pyocyanin, pyoverdin, or pyorubin, or both pyocyanin and pyoverdin, are easily recognized on the medium. Pyocyanin production is clearly distinguishable from other Pseudomonas pigments on MMA. In a comparative study, MMA identified 97% of the P. aeruginosa strains 24 h earlier than routine laboratory biochemical methods. Highly mucoid strains which did not produce detectable pigments on standard biochemicals produced detectable pigments on the MMA within 48 h. This medium can provide a very practical, reliable, and cost-effective means for early characterization of P. aeruginosa.

Topics: Culture Media; Cystic Fibrosis; Fermentation; Gram-Negative Bacteria; Humans; Lactose; Oligopeptides; Organic Chemicals; Phenazines; Pigments, Biological; Pseudomonas aeruginosa; Pyocyanine; Sputum