Microbiologic and clinical characteristics of biofilm-forming Candida parapsilosis isolates associated with fungaemia and their impact on mortality.

OBJECTIVES: Biofilm formation (BF) by fungal isolates may dramatically complicate infection. We determined the ability of Candida parapsilosis isolates from single fungaemia episodes to form biofilms and we analysed biofilm subgroups for antifungal susceptibility and pathogenic potential. We then correlated BF with clinical characteristics and outcomes of the episodes. METHODS: BF was measured using the crystal violet biomass assay. Antifungal susceptibility of preformed biofilms was assessed, and virulence was studied using the Galleria mellonella model. A retrospective analysis of patients' clinical records was performed. RESULTS: Of 190 patient-unique isolates, 84, 38 and 68 were identified as having high BF (HBF), moderate BF (MBF) or low BF (LBF), respectively. Among 30 randomly selected isolates, nine (eight HBF and one MBF), six (all HBF) and one (HBF) isolates had elevated sessile minimum inhibitory concentrations to fluconazole, anidulafungin or amphotericin B; all HBF and MBF isolates had elevated voriconazole sessile minimum inhibitory concentrations. G. mellonella killing rates of HBF isolates were significantly greater than MBF (or LBF) isolates (50% vs. 20%, 2 days from infection). By comparing HBF/MBF (106 patients) and LBF (84 patients) groups, we found that HBF/MBF patients had more central venous catheter-related fungaemias (62/106 (58.5%) vs. 29/84 (34.5%), p 0.001) and were more likely to die at 30 days from fungaemia onset (61/106 (57.5%) vs. 28/84 (33.3%), p 0.01). In the HBF/MBF group, azole antifungal therapy and central venous catheter removal were significantly associated with a higher and lower 30-day mortality rate, respectively. CONCLUSIONS: C. parapsilosis BF influences the clinical outcome in patients with fungaemia.

Clusters of patients with candidaemia due to genotypes of Candida albicans and Candida parapsilosis: differences in frequency between hospitals.

The presence of clusters (identical genotypes infecting different patients) suggests patient-to-patient transmission or a common source for strains. We report the results of a genotyping study based on microsatellite markers of Candida albicans (n = 179) and Candida parapsilosis (n = 76) causing candidaemia, to assess and compare the percentage of patients grouped in clusters during the study period (January 2010 to December 2012). The study was performed in two large tertiary hospitals in Madrid, Spain. We detected 145 C. albicans genotypes (21 in clusters) and 63 C. parapsilosis genotypes (seven in clusters). Clusters involved two to seven patients each. Most of the clusters in the two centres involved two patients for both species, but the number of patients included in each cluster differed between hospitals. Considering both species, the percentage of patients per cluster ranged from 19% to 38% (p < 0.05) in Hospital A and B respectively. Up to 2.9% of genotypes were present in both hospitals. Clusters of C. albicans and C. parapsilosis genotypes causing candidaemia differed between hospitals, suggesting differences in strain transmission. Occasionally, the same genotypes were found in patients admitted to different hospitals located in the same city.

Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Accurate species discrimination of filamentous fungi is essential, because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification through direct surface analysis of the fungal culture. By use of culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurements and MALDI BioTyper 2.0 software. The profiles of young and mature colonies were analysed for each of the reference strains, and species-specific spectral fingerprints were obtained. To evaluate the database, 103 blind-coded fungal isolates collected in the routine clinical microbiology laboratory were tested. As a reference method for species designation, multilocus sequencing was used. Eighty-five isolates were unequivocally identified to the species level (≥99% sequence similarity); 18 isolates producing ambiguous results at this threshold were initially rated as identified to the genus level only. Further molecular analysis definitively assigned these isolates to the species Aspergillus oryzae (17 isolates) and Aspergillus flavus (one isolate), concordant with the MALDI-TOF MS results. Excluding nine isolates that belong to the fungal species not included in our reference database, 91 (96.8%) of 94 isolates were identified by MALDI-TOF MS to the species level, in agreement with the results of the reference method; three isolates were identified to the genus level. In conclusion, MALDI-TOF MS is suitable for the routine identification of filamentous fungi in a medical microbiology laboratory.

Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in comparison to rpoB gene sequencing for species identification of bloodstream infection staphylococcal isolates.

As a result of variable expression of biochemical characters, misidentification by conventional phenotypic means often occurs with clinical isolates belonging to Staphylococcus species. Therefore, we evaluated the use of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of 450 blood isolates of the most relevant staphylococcal species, using sequence analysis of the rpoB gene as the reference method. A correct species identification by MALDI-TOF was obtained in 99.3% (447/450), with only three isolates being misidentified. In addition, MALDI-TOF correctly identified all the staphylococcal subspecies studied, including Staphylococcus capitis subsp. capitis and subsp. urealyticus, Staphylococcus cohnii subsp. urealyticus, Staphylococcus hominis subsp. novobiosepticus and subsp. hominis, Staphylococcus saprophyticus subsp. saprophyticus, Staphylococcus schleiferi subsp. schleiferi and Staphylococcus sciuri subsp. sciuri. Thus, MALDI-TOF MS-based species identification of staphylococci can be routinely achieved without any substantial costs for consumables or the time needed for labour-intensive DNA sequence analysis.

In vitro activity of Citrus bergamia (bergamot) oil against clinical isolates of dermatophytes.

OBJECTIVES: Recently, bergamot oil was shown to be a potent antifungal agent in vitro against clinically important Candida species. In this study, the activities of bergamot natural essence and its furocoumarin-free and distilled extracts on dermatophytes such as Trichophyton, Microsporum and Epidermophyton species were investigated. METHODS: In vitro susceptibility testing assays on 92 clinical isolates of dermatophytes (Trichophyton mentagrophytes n = 20, Trichophyton rubrum n = 18, Trichophyton interdigitale n = 15, Trichophyton tonsurans n = 2, Microsporum canis n = 24, Microsporum gypseum n = 1 and Epidermophyton floccosum n = 12) were performed using the CLSI M38-A broth microdilution method, except for employing an inoculum of 1-3 x 10(3) cfu/mL. MICs were determined at a visual endpoint reading of 80% inhibition compared with the growth control. RESULTS: MICs (v/v) of all fungi ranged from 0.156% to 2.5% for the natural essence, from 0.02% to 2.5% for the distilled extract, and from 0.08% to 1.25% for the furocoumarin-free extract. The three isolates of T. tonsurans and M. gypseum exhibited the highest MIC values. CONCLUSIONS: Data from this study indicate that bergamot oil is active in vitro against several common species of dermatophytes, suggesting its potential use for topical treatment of dermatophytoses.

Genetic diversity of bacterial strains isolated from soils, contaminated with polycyclic aromatic hydrocarbons, by 16S rRNA gene sequencing and amplified fragment length polymorphism fingerprinting.

In order to study microbial diversity in a polycyclic aromatic hydrocarbon-impacted soil, 14 bacterial strains were analyzed by 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis. Bacterial strains isolated from two different hydrocarbon-polluted sites were identified to the species level by 16S rRNA full-gene sequencing using MicroSeq 16S rRNA gene sequencing. Their genome was subsequently analyzed by high-resolution genotyping with AFLP analysis, in order to monitor species variability and to differentiate closely related strains. Cluster analysis based on AFLP fingerprinting showed intra-specific polymorphism, even among strains with 100% 16S rRNA gene sequence identity. The results show that AFLP is a powerful, highly reproducible and discriminatory tool for revealing genetic relationships in bacterial populations. The ability to differentiate and track related closely microbes is fundamental for studying structure and dynamics of microbial communities in contaminated ecosystems.

Validation of a pXO2-A PCR assay to explore diversity among Italian isolates of Bacillus anthracis strains closely related to the live, attenuated Carbosap vaccine.

Several circulating Bacillus anthracis strains isolated in Italy and belonging to the A1.a cluster, genotype 3 (A1.a-3) are genotypically indistinguishable from Carbosap, a live attenuated vaccine strain, containing both pXO1 and pXO2 plasmids. The genotype was assessed by using eight-locus multilocus variable-number tandem repeat analysis. We describe here the use of a ninth locus able to explore variability among strains that have the same genotype. It is important to be able to genotype the wild isolate of B. anthracis strains from outbreaks of anthrax in areas where Carbosap vaccination of cattle and sheep is common practice. A total of 27 representative field strains isolated in Italy and four vaccinal strains, namely, Carbosap, Sterne, Pasteur I, and Pasteur II, were characterized by a ninth marker, called pXO2-A. Twenty-three field strains were genotype 3 and therefore identical to Carbosap. The marker was in the pXO2 plasmid and is based on the polymorphism of the already-known VX2-3 locus. Detection was obtained by PCR with fluorescence-labeled forward primers in order to produce appropriate fragments for capillary electrophoresis with an ABI 310 genetic analyzer. Genetic relationships showed heterogeneity in all of the examined samples. Interestingly, with respect to genotype 3, samples grouped into eight different subtypes, A to H, and the subtype G, had only two samples indistinguishable from Carbosap. The results of the present study confirm the validity of a hierarchical progressive protocol for discrimination among closely related isolates.

Chlamydia species as a cause of community-acquired pneumonia in Canada.

Chlamydia pneumoniae has been implicated as a cause of community-acquired pneumonia (CAP) in several studies. However, there has been no comprehensive study of the role of Chlamydia species (C. pneumoniae, C. psittaci (avian and feline strains) and C. pecorum) as a cause of CAP. The aim of the present study was to determine the role of C. pneumoniae, C. psittaci and C. pecorum as causes of CAP. A prospective cohort observational study of CAP was conducted at 15 teaching centres in eight Canadian provinces between January 1996-October 1997. Acute (n=539) and convalescent (n=272) serum samples were obtained for determination of antibody titres to C. pneumoniae, C. psittaci, C. pecorum, C. trachomatis, Mycoplasma pneumoniae, Legionella pneumophila serogroups I-VI, Streptococcus pneumoniae and various respiratory viruses. Twelve of 539 (2.2%) patients had acute C. pneumoniae pneumonia and an additional 32 (5.9%) had possible acute infection. C. pneumoniae was the sole pathogen in 16 of 42 (38.1%) of these patients. The most common copathogens were S. pneumoniae, respiratory syncytial virus and influenza virus type A. C. pneumoniae pneumonia patients were older and more likely to show congestive heart failure compared to bacteraemic S. pneumoniae patients. The latter had a lower mean diastolic blood pressure, a higher white blood cell count and a lower arterial carbon dioxide tension. Two patients had antibody titres suggestive of recent infection with the feline strain of C. psittaci. Although numerically Chlamydia pneumoniae is an important cause of community-acquired pneumonia, no distinctive clinical features associated with this pathogen were detected in the present study. Feline Chlamydia psittaci may cause a few cases of community-acquired pneumonia. Avian Chlamydia psittaci should be considered only if there is a compatible epidemiological history.

Legionella-like and other amoebal pathogens as agents of community-acquired pneumonia.

We tested serum specimens from three groups of patients with pneumonia by indirect immunofluorescence against Legionella-like amoebal pathogens (LLAPs) 1-7, 9, 10, 12, 13; Parachlamydia acanthamoeba strains BN 9 and Hall's coccus; and Afipia felis. We found that LLAPs play a role (albeit an infrequent one) in community-acquired pneumonia, usually as a co-pathogen but sometimes as the sole identified pathogen.

Comparative activity of new quinolones against 326 clinical isolates of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an important emerging pathogen causing a variety of infections in severely ill patients. This microorganism is inherently resistant to many antibiotics, and only a few therapeutic options are available. The principal aim of this study was to assess the in vitro activity of new quinolones against this pathogen. Three hundred and twenty-six single clinical isolates were tested in this study. The MIC(90) was 16 mg/L for ciprofloxacin, 8 mg/L for levofloxacin and gatifloxacin, 4 mg/L for trovafloxacin, moxifloxacin and sparfloxacin and 2 mg/L for clinafloxacin. At a 2 mg/L concentration, a C(max) lung:MIC ratio of >/=10 can be reached for 95%, 84.3%, 83.1% and 81.5% of isolates, respectively, for clinafloxacin, trovafloxacin, moxifloxacin and sparfloxacin (P < 0. 001 compared with levofloxacin and ciprofloxacin). In spite of the rare but serious adverse events associated with the new-generation quinolones, these agents may become very useful in the treatment of certain severe or life-threatening infectious conditions due to S. maltophilia, notably lower respiratory tract infections.

Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don.

A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the 4-hydroxylation of desacetoxyvindoline was purified to homogeneity. Three oligopeptides isolated from a tryptic digest of the purified protein were microsequenced and one oligopeptide showed significant homology to hyoscyamine 6 beta-hydroxylase from Hyoscyamus niger. A 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a Catharanthus roseus cDNA library and three clones, cD4H-1 to -3, were isolated. Although none of the three clones were full-length, the open reading frame on each clone encoded a putative protein containing the sequence of all three peptides. Primer extension analysis suggested that cD4H-3, the longest cDNA clone, was missing 156 bp at the 5' end of the clone and sequencing of the genomic clone, gD4H-8, confirmed these results. Southern blot analysis suggested that d4h is present as a single-copy gene in C. roseus which is a diploid plant, and the significant differences in the sequence of the 3'-UTR between cD4H-1 and -3 suggest that they represent dimorphic alleles of the same hydroxylase. The identity of the clone was further confirmed when extracts of transformed Escherichia coli expressed D4H enzyme activity. The D4H clone encoded a putative protein of 401 amino acids with a calculated molecular mass of 45.5 kDa and the amino acid sequence showed a high degree of similarity with those of a growing family of 2-oxoglutarate-dependent dioxygenases of plant and fungal origin. The similarity was not restricted to the dioxygenase protein sequences but was also extended to the gene structure and organization since the 205 and 1720 bp introns of d4h were inserted around the same highly conserved amino acid consensus sequences as those for e8 protein, hyoscyamine-6 beta-hydroxylase and ethylene-forming enzyme. These results provide further support that a common ancestral gene is responsible for the appearance of this family of dioxygenases. Hydroxylase assays and RNA blot hybridization studies showed that enzyme activity followed closely the levels of d4h transcripts, occurring predominantly in young leaves and in much lower levels in stems and fruits. In contrast, etiolated seedlings which contained considerable levels of d4h transcripts had almost undetectable hydroxylase activity, whereas exposure of seedlings to light resulted in a rapid increase of enzyme activity without a significant further increase in d4h transcripts over those detected in dark-grown seedlings. These results suggest that the activating effect of light may occur at a point downstream of transcription which remains to be elucidated.

Oxidative inactivation of human 5-lipoxygenase in phosphatidylcholine vesicles.

Human 5-lipoxygenase is a non-heme iron protein which possesses 5-oxygenase, leukotriene A4 synthase and pseudoperoxidase activities and which undergoes a rapid irreversible inactivation during these reactions. The inactivation of the enzyme was dependent on the structural characteristics of the substrate for the reaction, on O2 concentration and on exposure to phospholipids and calcium. The apparent first-order rate constant for enzyme inactivation (k(in)) was 0.6 min(-1) during the oxygenation of arachidonic acid in air-saturated buffer containing phosphatidylcholine vesicles and Ca2+. The rate of enzyme inactivation was dependent on the substrate for the reaction and was about threefold slower during the oxygenation of 5,8-icosadienoic acid and 12(S)-hydroxyicosatetraenoic acid compared with arachidonic acid. Lowering the 02 concentration to 60 microM during the oxygenation of arachidonic acid also caused a 2.5-fold decrease in k(in) without affecting the initial rate of the reaction resulting in an increase in both 5-hydroperoxyicosatetraenoic acid (5-HPETE) and leukotriene A4 accumulation. The concentration of 02 for half-maximal activity (initial rate and product accumulation) was approximately 10 microM. In contrast, the activity and the rate of inactivation during the leukotriene A4 synthase reaction with exogenous 5-HPETE (k(in)=2.0 min(-1) were independent of 02 concentration. A rapid inactivation of the enzyme was also observed during aerobic incubation with phosphatidylcholine vesicles and Ca2+ in the absence of substrate, with a sequential loss of the oxygenase (t1/2 = 0.5 min) and pseudoperoxidase (t1/2 = 7 min) activities. Protection against this turnover-independent inactivation was observed in the presence of the selective reversible 5-lipoxygenase inhibitor L-739,010 ([1S, 5R] 3-cyano-1-(3-furyl)-6-(6-[3-(3 alpha-hydroxy-6,8-dioxyabicyclo [3.2.11 octanyl)] pyridin-2-ylmethoxy) naphthalene) and by prior treatment of vesicles with sodium borohydride and, to a lesser extent, by glutathione peroxidase. The results show that the inactivation of 5-lipoxygenase in phospholipid vesicles is dependent on the structure of the unsaturated fatty acid substrate for the reaction, on the concentration of oxygen and on a turnover-independent oxidation at the active-site leading to the sequential loss of the oxygenase and pseudoperoxidase activities of the enzyme.

2-oxoglutarate-dependent dioxygenase and related enzymes: biochemical characterization.

Hydroxylation reactions are catalysed by a few major subclasses of enzymes which are ubiquitously distributed in nature. Dioxygenases generally occur as soluble enzymes where they catalyse a diversity of oxygenation reactions in a large number of metabolic pathways in animals, plants and micro-organisms. This review discusses recent advances in the biochemistry and molecular biology of dioxygenases occurring in different biological systems.

Purification, characterization, and kinetic analysis of a 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis from Catharanthus roseus.

A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the hydroxylation at position 4 of the indole alkaloid, desacetoxyvindoline has been purified to near homogeneity from Catharanthus roseus. The purification procedure combined conventional chromatographic methods and cosubstrate affinity chromatography on alpha-ketoglutarate-Sepharose. The specific activity of the 4-hydroxylase was enriched over 2000-fold compared to the crude homogenate with a recovery of 1.6%. The molecular mass of the native and denatured 4-hydroxylase was found to be 45 and 44.7 kDa, respectively, suggesting that the native enzyme is a monomer. Two-dimensional isoelectric focusing under denaturing conditions resolved the purified 4-hydroxylase into three charge isoforms of pI values 4.6, 4.7, and 4.8. The enzyme did not require most divalent cations, but inactive enzyme was reactivated in a time-dependent manner by incubation with ferrous ions. The mechanism of action of desacetoxyvindoline 4-hydroxylase was investigated. The results of substrate interaction kinetics and product inhibition studies suggest an Ordered Ter Ter mechanism where 2-oxoglutarate is the first substrate to bind followed by the binding of O2 and desacetoxyvindoline. The first product to be released was deacetylvindoline followed by CO2 and succinate, respectively.

Isolation and Characterization of a 2-Oxoglutarate Dependent Dioxygenase Involved in the Second-to-Last Step in Vindoline Biosynthesis.

Young leaves from Catharanthus roseus plants contain the enzymes which convert the monoterpenoid indole alkaloid, tabersonine by three hydroxylations, two methylations, and one acetylation step to vindoline. A novel direct enzyme assay has been developed for a hydroxylase involved in vindoline biosynthesis, which catalyzes the C4-hydroxylation of 2,3-dihydro-3-hydroxy-N(1)-methyltabersonine to the 3,4-dihydroxy derivative. The enzyme showed an absolute requirement for 2-oxoglutarate and enzymatic activity was enhanced by ascorbate, establishing it as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). The hydroxylase exhibited specificity for position 4 of various alkaloid substrates. The enzyme exhibited a pH optima between 7 and 8 and an apparent molecular weight of 45,000. The appearance of 4-hydroxylase activity was developmentally regulated and was shown to be inducible by light treatment of seedlings. Substrate specificity studies of this enzyme for indole alkaloid substrate suggested that hydroxylation at position 3 and N-methylation occur prior to hydroxylation at position 4. This is in agreement with previous studies which suggest that C4-hydroxylation is the second to last step in vindoline biosynthesis in Catharanthus roseus.