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Results for “Automated phenotyping”

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PubMed1998

Detection of methicillin resistance in coagulase-negative staphylococci initially reported as methicillin susceptible using automated methods.

Reliable detection of methicillin resistance in coagulase-negative staphylococci (CNS) is required for appropriate therapy of serious infections from these pathogens. To determine the most accurate method of measuring methicillin resistance in CNS initially reported as methicillin susceptible by automated methods, we compared mecA detection by polymerase chain reaction (PCR) with phenotypic methods. One hundred eighty-eight blood culture isolates of CNS that were initially reported as susceptible to methicillin using commercial methods (Vitek or MicroScan) were tested by agar dilution, disk diffusion, oxacillin salt agar screen plate, and a multiplex PCR assay using primer sets for mecA and 16S rRNA. Sixteen isolates (8.5%) previously reported as methicillin susceptible by automated methods contained the mecA gene. MICs of these isolates ranged from 0.5 microgram/mL to > or = 128 micrograms/mL. Ten of these isolates had MICs equal to or below the NCCLS breakpoint of 2 micrograms/mL. Six of the 10 isolates (4 with MICs of 0.5 microgram/mL and 2 with MICs of 2 micrograms/mL) did not grow on any of the oxacillin screen plates after 48 h of incubation at 30 degrees C or 35 degrees C. All six isolates were induced to grow in the presence of oxacillin at 128 micrograms/mL by serial passaging on plates containing increasing concentrations of antibiotic. Retesting with MicroScan and Vitek detected methicillin resistance in 7 and 10 isolates, respectively. Disk diffusion testing with incubation for 48 h proved to be the next best method after PCR for detection of methicillin resistance (15 of 16 isolates). Commercial automated methods and some methods recommended by National Committee for Clinical Laboratory Standards may not detect methicillin resistance in CNS that carry the mecA gene and have MICs just below breakpoint.

Bacterial Proteins↗

PubMed2006

Expanding the COL7A1 mutation database: novel and recurrent mutations and unusual genotype-phenotype constellations in 41 patients with dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB), a heterogeneous hereditary skin disorder characterized by trauma-induced blistering and scarring, affects thousands of families worldwide. The clinical manifestations extend from minor nail dystrophy to severe life-threatening blistering, making early molecular diagnosis and prognostication of utmost importance for the affected families. DEB is caused by mutations in the COL7A1 gene encoding collagen VII in the skin. Molecular diagnostics and genotype-phenotype correlations in DEB remain complex owing to the gene structure, large variety of mutations, high rate of novel mutations, complex protein structure and assembly, and the heterogeneity of phenotypes. Here, we report an efficient strategy for COL7A1 mutation detection using direct automated DNA sequencing and implementation of software tools. With this approach, COL7A1 mutations of 41 DEB families were disclosed. Twenty-four mutations were novel and two recurrent. Elucidation of biological consequences of the mutations helped define disease mechanisms, but also revealed several unusual genotypic and/or phenotypic constellations, which impeded the diagnostics and prognostication. In addition, the studies disclosed a de novo mutation in recessive DEB and two new polymorphisms in the COL7A1 gene.

Collagen Type VII↗

PubMed2006

Properties of voltage-gated Na+ channels in the human rhabdomyosarcoma cell-line SJ-RH30: conventional and automated patch clamp analysis.

Conventional and automated patch clamp electrophysiology were used to characterise the Na+ current of the SJ-RH30 human rhabdomyosarcoma. In conventional recordings SJ-RH30 cells exhibited a fast activating, fast inactivating Na+ current at potentials positive to -40 mV; in full current-voltage curves maximum current occurred between -20 and -10 mV. Inactivation kinetics at 0 mV were biexponential with time constants of 0.5 and 3.7 ms. Deinactivation at -90 mV also exhibited two kinetic components. Tetrodotoxin (TTX) blocked the Na+ current completely at 1 microM. The NaV 1.4 selective toxin mu-CTx-GIIIB reversibly blocked the Na+ current approximately 60% at 10 microM. Very similar biophysical behaviour was observed in automated patch clamp and conventional recordings. For example, inactivation mid-point was -72+/-2 mV (slope factor 7.2+/-0.2) in automated patch clamp and -74+/-2 mV (slope factor 7.4+/-0.4) with conventional recording. The corresponding values for activation mid-point were -33.2+/-2.4 and -30.3+/-2.7 mV (slope 5.8+/-0.3 and 6.4+/-0.3, respectively). The throughput of the automated method was used to generate additional pharmacological data on inhibition of the Na+ current. TTX inhibited with an IC50 of 23 nM. Mu-CTx-GIIIB also inhibited the channel in a concentration-dependent manner. Inhibition produced by both tetracaine and amitriptyline were shown to be frequency-dependent. Our experiments indicate that the Na+ current of SJ-RH30 cells arises mainly from channels with a phenotype like recombinant NaV 1.4 channels. The suitability of these cells for automated patch clamp suggests they may be useful for higher throughput studies of the interaction of drugs with human skeletal muscle Na+ channels.

Action Potentials↗

PubMedDate not supplied

Heterogeneity of the phenotype Jk(a-b-) found in Japanese.

Fourteen Jk(a-b-) persons were detected by testing 638,460 Osaka blood donors with an automated 2 M urea technique. Two of these 14 Jk(a-b-) samples were quite different from the hitherto reported Jk(a-b-) phenotype, and a family study showed that the mode of the inheritance was dominant. The red cell membranes of these Jk(a-b-) samples were studied by polyacrylamide gel electrophoresis and unusual protein bands (apparent mw, 67,000 d) were detected.

Blood Group Antigens↗

PubMed2005

An automated system for measuring parameters of nematode sinusoidal movement.

BACKGROUND: Nematode sinusoidal movement has been used as a phenotype in many studies of C. elegans development, behavior and physiology. A thorough understanding of the ways in which genes control these aspects of biology depends, in part, on the accuracy of phenotypic analysis. While worms that move poorly are relatively easy to describe, description of hyperactive movement and movement modulation presents more of a challenge. An enhanced capability to analyze all the complexities of nematode movement will thus help our understanding of how genes control behavior. RESULTS: We have developed a user-friendly system to analyze nematode movement in an automated and quantitative manner. In this system nematodes are automatically recognized and a computer-controlled microscope stage ensures that the nematode is kept within the camera field of view while video images from the camera are stored on videotape. In a second step, the images from the videotapes are processed to recognize the worm and to extract its changing position and posture over time. From this information, a variety of movement parameters are calculated. These parameters include the velocity of the worm's centroid, the velocity of the worm along its track, the extent and frequency of body bending, the amplitude and wavelength of the sinusoidal movement, and the propagation of the contraction wave along the body. The length of the worm is also determined and used to normalize the amplitude and wavelength measurements. To demonstrate the utility of this system, we report here a comparison of movement parameters for a small set of mutants affecting the Go/Gq mediated signaling network that controls acetylcholine release at the neuromuscular junction. The system allows comparison of distinct genotypes that affect movement similarly (activation of Gq-alpha versus loss of Go-alpha function), as well as of different mutant alleles at a single locus (null and dominant negative alleles of the goa-1 gene, which encodes Go-alpha). We also demonstrate the use of this system for analyzing the effects of toxic agents. Concentration-response curves for the toxicants arsenite and aldicarb, both of which affect motility, were determined for wild-type and several mutant strains, identifying P-glycoprotein mutants as not significantly more sensitive to either compound, while cat-4 mutants are more sensitive to arsenite but not aldicarb. CONCLUSIONS: Automated analysis of nematode movement facilitates a broad spectrum of experiments. Detailed genetic analysis of multiple alleles and of distinct genes in a regulatory network is now possible. These studies will facilitate quantitative modeling of C. elegans movement, as well as a comparison of gene function. Concentration-response curves will allow rigorous analysis of toxic agents as well as of pharmacological agents. This type of system thus represents a powerful analytical tool that can be readily coupled with the molecular genetics of nematodes.

Acetylcholine↗

PubMed2001

Mutation analysis of the HFE gene in German hemochromatosis patients and controls using automated SSCP-based capillary electrophoresis and a new PCR-ELISA technique.

BACKGROUND: The diagnosis of hereditary hemochromatosis (HH) before the onset of iron-overload has been difficult in the past. However, a convincing candidate gene for HH: HFE has been described recently. The aims of this study were: 1) To determine the prevalence of the hemochromatosis associated mutations C282Y and H63D of the HFE gene in patients from Southern Germany with hemochromatosis phenotype; and 2) to test two new, time- and cost-saving methods: automated SSCP-based capillary electrophoresis (SSCP-CE) and a PCR-ELISA technique for the analysis of HFE mutations. METHODS: HFE genotype was studied in 36 unrelated HH patients and 126 controls from Southern Germany. In addition, family screening was performed in 76 relatives. The C282Y and H63D mutations were detected using SSCP-CE and restriction length polymorphism (RFLP). The C282Y mutation was additionally analysed by a PCR-ELISA. RESULTS: Twenty-six (72%) HH patients were homozygous for mutation C282Y, and three compound heterozygous for C282Y and H63D. One patient was homozygous for H63D. By performing family screening, six additional patients with the +/+ C282Y mutation were identified. The results of the SSCP-CE and the PCR-ELISA analysis agreed completely with data obtained by RFLP. CONCLUSIONS: SSCP-CE and PCR-ELISA analysis proved to be reliable methods for HFE genotyping and therefore represent cost- and time-effective alternative methods to the widely used restriction analysis allowing large populations to be screened for HH associated with HFE mutations. Surprisingly, only 72% of our HH patients had the C282Y +/+ genotype. This indicates that hemochromatosis in Southern Germany is genetically more heterogeneous than in other regions. A challenge for the future will be to define the genetic or environmental factors responsible for iron-overload in HH patients who do not show typical alterations of the HFE gene.

DNA Mutational Analysis↗

PubMed1999

A new structurally dissolvable self-reproducing loop evolving in a simple cellular automata space.

We constructed a simple evolutionary system, "evoloop," on a deterministic nine-state five-neighbor cellular automata (CA) space by improving the structurally dissolvable self-reproducing loop we had previously contrived [14] after Langton's self-reproducing loop [7]. The principal role of this improvement is to enhance the adaptability (a degree of the variety of situations in which structures in the CA space can operate regularly) of the self-reproductive mechanism of loops. The experiment with evoloop met with the intriguing result that, though no mechanism was explicitly provided to promote evolution, the loops varied through direct interaction of their phenotypes, smaller individuals were naturally selected thanks to their quicker self-reproductive ability, and the whole population gradually evolved toward the smallest ones. This result gives a unique example of evolution of self-replicators where genotypical variation is caused by precedent phenotypical variation. Such interrelation of genotype and phenotype would be one of the important factors driving the evolutionary process of primitive life forms that might have actually occurred in ancient times.

Artificial Intelligence↗

PubMed2006

Phenotypes of Drosophila brain neurons in primary culture reveal a role for fascin in neurite shape and trajectory.

Subtle cellular phenotypes in the CNS may evade detection by routine histopathology. Here, we demonstrate the value of primary culture for revealing genetically determined neuronal phenotypes at high resolution. Gamma neurons of Drosophila melanogaster mushroom bodies (MBs) are remodeled during metamorphosis under the control of the steroid hormone 20-hydroxyecdysone (20E). In vitro, wild-type gamma neurons retain characteristic morphogenetic features, notably a single axon-like dominant primary process and an arbor of short dendrite-like processes, as determined with microtubule-polarity markers. We found three distinct genetically determined phenotypes of cultured neurons from grossly normal brains, suggesting that subtle in vivo attributes are unmasked and amplified in vitro. First, the neurite outgrowth response to 20E is sexually dimorphic, being much greater in female than in male gamma neurons. Second, the gamma neuron-specific "naked runt" phenotype results from transgenic insertion of an MB-specific promoter. Third, the recessive, pan-neuronal "filagree" phenotype maps to singed, which encodes the actin-bundling protein fascin. Fascin deficiency does not impair the 20E response, but neurites fail to maintain their normal, nearly straight trajectory, instead forming curls and hooks. This is accompanied by abnormally distributed filamentous actin. This is the first demonstration of fascin function in neuronal morphogenesis. Our findings, along with the regulation of human Fascin1 (OMIM 602689) by CREB (cAMP response element-binding protein) binding protein, suggest FSCN1 as a candidate gene for developmental brain disorders. We developed an automated method of computing neurite curvature and classifying neurons based on curvature phenotype. This will facilitate detection of genetic and pharmacological modifiers of neuronal defects resulting from fascin deficiency.

Actins↗

PubMed2001

K, Fy(a), and Jk(a) phenotyping of donor RBCs on microplates.

BACKGROUND: In many cases, the search for compatible blood for patients with clinically significant RBC alloantibodies is difficult and time-consuming. To date, it has been considered necessary only to phenotype the blood donors for ABO group and D. There has been long experience with automated routine analysis (ABO, C, c, D, E, and e typing and RBC antibody screening), using robotic dispensers and computerized interpretation of microplate results. The purpose of this study was to explore the possibilities of also phenotyping for K, Fy(a), and Jk(a), as antibodies directed against these antigens (together with Rh antigens) are the most common clinically significant alloantibodies in the Swedish population. STUDY DESIGN AND METHODS: One thousand thirty-one EDTA samples from blood donors were phenotyped for K, Fy(a), and Jk(a) by use of an IAT with PEG on microplates. The findings were compared to those using conventional IAT in tube's and the microcolumn gel test (DiaMed-ID, DiaMed). RESULTS: All typing results with the microplate method were correct. All reactions for K and Fy(a) typing could be interpreted by the computer. The results for Jk(a) were indeterminate in 1.4 percent (14/1031) of the samples. CONCLUSION: The PEG-IAT microplate method gave reliable results that were suitable for routine phenotyping, thus making available a stock of phenotyped blood at reasonable cost, ready for delivery when required.

Blood Donors↗

PubMed2004

Blood coagulation kinetics: high throughput method for real-time reaction monitoring.

A high throughput 384-well plate assay of blood function in 60 microl reactions with the fluorogenic thrombin substrate, boc-VPR-MCA, allowed for real-time monitoring of coagulation under a diverse set of reaction conditions. Using recalcified, citrated whole blood diluted 3-fold with corn trypsin inhibitor (to block Factor XIIa), addition of 0 to 13.8 pM of tissue factor (TF) reduced the time of maximal rate of thrombin production T(max) from 45 min to 11 min. Over this range of TF,T(max) was reduced from 35 min to 6 min by co-addition of 10 nM convulxin to activate platelets via GPVI. The maximal rate of thrombin production at T(max) was not a function of exogenously-added TF,Va, or reVIIa, but increased 30% with added convulxin. Addition of 0.07 to 0.7 pM TF along with convulxin produced small, but detectable reductions in T(max). Addition of up to 0.67 nM reVIIa reduced T(max) by up to 53% in the range of 0.7 to 7 pM TF. Interestingly, platelet factor 4 (2.7 microM) caused a prolongation of T(max) from 45 min to 78 min at 0 TF, while protamine (1.8 microM) reduced T(max) to 30 min at 0 TF. Finally, combinatorial reaction studies with exogenously-added ADP, histamine, fMLP, indomethacin, anti-CD18, and fibrinogen revealed no unusual synergies amongst the agents, but demonstrated a striking procoagulant activity of added fibrinogen, due to protease contaminants in the "purified" fibrinogen. This high throughput approach allowed automated profiling of blood (50 reactions/ml of blood) to generate large data sets for testing cellular-proteomic kinetic models, screening drug interactions, and potentially monitoring subtle changes in the functional phenotype of a patient blood sample.

Automation↗

PubMed1999

Computer-assisted morphometry of cell-substratum contacts.

AIM: Quantitative analysis of size and shape of the cell-substratum contacts in Dictyostelium and comparison of these parameters between wild-type cells and the cells bearing cytoskeletal protein mutations. METHODS: Reflection interference contrast microscopy (RICM) was used to image the areas of contact between aggregation-competent Dictyostelium cells and weakly adhesive mica surfaces. The cell-substratum contact areas were automatically identified in RICM micrographs by digital image processing. Information about the size and shape of the contact areas was obtained by using the shape descriptors based on two-dimensional geometrical moment invariants. RESULTS: Lack of either of the two actin-crosslinking proteins, a-actinin and 120 kDa gelation factor, similarly affects the cell-substratum interactions of Dictyostelium cells. The shape descriptors, elongation and dispersion, of the contact areas were reduced by 10% to 30% in mutant cells when compared to the wild type, but the size of the contacts was not affected. CONCLUSION: Video microscopy combined with digital image processing and quantitative image analysis is capable of revealing small phenotypic effects of cytoskeletal protein mutations on the level of single cells. Such automated microscopic methods are expected to gain importance and find a widespread use in biomedicine.

Actinin↗

PubMed2005

Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci.

To compare commonly used phenotypic methods with genotypic identification methods 47 clinical isolates of coagulase-negative staphylococci (CONS), 10 CONS ATCC strains, and a Staphylococcus aureus clinical isolate were identified using the API Staph ID test, BD Phoenix Automated Microbiology System, and 16S rRNA gene and tuf gene sequencing. When necessary part of the sodA gene was sequenced for definitive identification. The results show that tuf gene sequencing is the best method for identification of CONS, but the API Staph ID test is a reasonably reliable phenotypic alternative. The performance of the BD Phoenix Automated Microbiology System for identification of CONS is poor. The present study also showed that although genotypic methods are clearly superior to phenotypic identifications, a drawback of sequence-based genotypic methods may be a lack of quality of deposited sequences in data banks. In particular, 16S rRNA gene sequencing suffers from the lack of high quality among sequences deposited in GenBank. Furthermore, genotypic identification based on 16S rRNA sequences has limited discriminating power for closely related Staphylococcus species. We propose partial sequencing of the tuf gene as a reliable and reproducible method for identification of CONS species.

Base Sequence↗

PubMed2000

Description of a new mutation in rhodopsin, Pro23Ala, and comparison with electroretinographic and clinical characteristics of the Pro23His mutation.

OBJECTIVES: To report the clinical characteristics of a family with autosomal dominant retinitis pigmentosa caused by a proline-to-alanine mutation at codon 23 (Pro23Ala) of the rhodopsin gene and to compare this phenotype with that associated with the more common proline-to-histidine mutation at codon 23 (Pro23His). METHODS: We examined 6 patients within a single pedigree. The electroretinograms (ERGs) of 35 patients with known Pro23His mutations and of 22 healthy individuals were reviewed. Scotopic dim flash-response amplitudes, maximum combined-response amplitudes, and photopic-response amplitudes from the ERGs of these patients were plotted against age. The ERG indices of 5 individuals in the Pro23Ala family were compared with those of the patients with Pro23His mutations and of healthy individuals. Multiple linear regression was performed to evaluate the effect of age and mutation type on amplitudes. Mutation detection was performed using single-strand conformation polymorphism analysis, followed by automated DNA sequencing. RESULTS: Patients with the Pro23Ala mutation have a clinical phenotype characterized by onset of symptoms in the second to fourth decades of life, loss of superior visual field with relatively well-preserved inferior fields, and mild nyctalopia. Comparison with patients with the Pro23His mutation demonstrates statistically significant differences (P<.001) in responses to dim flash, maximum combined, and photopic responses between patients with these mutations after controlling for the effects of age. Patients with Pro23Ala mutations were less affected by ERG criteria than patients with Pro23His mutations. Patients with Pro23Ala mutations also differed significantly from healthy patients in all ERG indices examined (P<.001), after controlling for age. CONCLUSION: We describe a rare mutation in codon 23 of rhodopsin causing autosomal dominant retinitis pigmentosa. The retinal dystrophy associated with the Pro23Ala mutation is characteristically mild in presentation and course, with greater preservation of ERG amplitudes than the more prevalent Pro23His mutation. Arch Ophthalmol. 2000;118:1269-1276

Adolescent↗

PubMed2005

Computerized image analysis for quantitative neuronal phenotyping in zebrafish.

An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimer's disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies.

Animals↗

PubMed2001

Molecular differentiation of Lactococcus lactis subspecies lactis and cremoris strains by ribotyping and site specific-PCR.

Twenty-five strains of Lactococcus lactis subspecies lactis and subspecies cremoris obtained from dairy industry and environmental collections were examined by 16S RNA automated ribotyping profiles and site-specific PCR (S-PCR). By automated ribotyping, the majority of strains were classified in accordance with phenotypic characterization, with the exception of one lactis (220) and two cremoris (BO32 and 140) strains. A complete differentiation of subspecies lactis and cremoris in agreement with conventional phenotypic methods was achieved by S-PCR with a set of site-specific primer pairs (PR1, RM4, and F3) designed particularly from a deletion region found in subspecies cremoris, but not in lactis. Therefore, S-PCR with primers (PR1, RM4, and F3) is a rapid and very sensitive method for the distinction of lactis and cremoris subspecies in dairy production.

DNA Fingerprinting↗

PubMed2005

Gene mutations in retinitis pigmentosa and their clinical implications.

Retinitis pigmentosa (RP) is a group of inherited progressive retinal diseases affecting about 1 in 3500 people worldwide. So far, there is no prevention or cure, with permanent visual loss or even blindness the ultimate consequence usually after midlife. The genetics of RP are complex. It can be sporadic, autosomal dominant, autosomal recessive, or X-linked. Thirty-two genes are known to be associated with RP, sometimes the same gene gets involved in different inheritance traits. Some RP cases have a digenic cause. About 60% RP cases still have no known genetic cause. A large number of mutations cause RP, and they can be deletions, insertions, or substitutions that cause missense mutations or truncations. The RHO, RP1, and RPGR genes contribute the greatest number of known mutations causative of RP. But there is no single mutation that alone accounts for more than 10% of unrelated patients. Genetic testing for RP therefore requires screening for a group of genes. High-throughput and automated sequence detection technologies are essential. Due to the complexity in phenotype and genetics, and the fact that RP is untreatable, genetic testing for presymptomatic diagnosis of RP is controversial. Meanwhile, new genes are still to be identified, mostly by family linkage and sib-pair analysis. Research on gene therapy for RP requires information on gene mutations causative of RP.

Eye Proteins↗

PubMed1996

Development of a PCR-based diagnostic assay for the determination of KEL genotype in donor blood samples.

The polymorphism which determines expression of Kell and Cellano antigens on the redcell surface has been reported to be a single C-->T nucleotide substitution at residue 701 where T codes for the presence of Kell antigen. This was confirmed by the direct automated sequencing of PCR products amplified from individuals of known Kell phenotype. The substitution creates a Bsm I restriction enzyme site and this has formed the basis for the development of a PCR-based diagnostic assay for the determination of Kell phenotype in samples of donor blood. The assay is based on RFLP analysis of coamplified PCR products, one of which spans the K/k polymorphic site, and one control fragment which contains a Bsm I site. Digestion of the PCR products with Bsm I restriction enzyme and subsequent gel analysis of the digest allowed unequivocal determination of the K/k status of all of the samples tested.

Blood Donors↗

PubMed2006

Microbiology of airway disease in a cohort of patients with cystic fibrosis.

BACKGROUND: Recent reports document an increasing incidence of new Gram-negative pathogens such as Stenotrophomonas maltophilia and Alcaligenes xylosoxidans isolated from patients with Cystic Fibrosis, along with an increase in common Gram-negative pathogens such as Pseudomonas aeruginosa and Burkholderia cepacia complex. Furthermore, the increase in multidrug-resistance of such organisms makes the therapeutic management of these patients more problematic. Therefore, careful isolation and identification, and accurate studies of susceptibility to antibiotics are critical for predicting the spread of strains, improving therapeutic measures and facilitating our understanding of the epidemiology of emerging pathogens. The first aim of this study was to determine the incidence and the prevalence of colonization by Gram-negative organisms isolated from respiratory samples of Cystic Fibrosis patients in the Regional Referral Cystic Fibrosis Centre of Naples; the second was to evaluate the spectrum of multidrug-resistance of these organisms. METHODS: Patients (n = 300) attending the Regional Cystic Fibrosis Unit were enrolled in this study over 3 years. Sputum was processed for microscopic tests and culture. An automated system, Phoenix (Becton Dickinson, Sparks, Maryland, USA), was used for phenotypic identification of all strains; the API 20 NE identification system (bioMérieux, Marcy l'Etoile, France) was used when the identification with the Phoenix system was inaccurate. A PCR-RFLP method was used to characterize the organisms in the Burkholderia cepacia complex. A chemosusceptibility test on microbroth dilutions (Phoenix) was used. Primary outcomes such as FEV1 were correlate with different pathogens. RESULTS: During the period of study, 40% of patients was infected by Pseudomonas aeruginosa, 7% by Burkholderia cepacia complex, 11% by Stenotrophomonas maltophilia and 7% by Alcaligenes xylosoxidans. Of the strains isolated, 460 were multidrug-resistant. Multiresistant were Pseudomonas aeruginosa and Burkholderia cepacia complex. CONCLUSION: The results confirm previously reported data; in particular, they show an increase the isolation of non-fermentative Gram-negative bacteria in Cystic Fibrosis patients. They also demonstrate increased resistance to antibiotics. Beta-lactams are rarely effective, with exception of ceftazidime, which is the most efficacious agent against multiresistant strains. Aminoglycosides and quinolones are poorly efficacious.

Adolescent↗