Outer membrane vesicles produced by Burkholderia cepacia cultured with subinhibitory concentrations of ceftazidime enhance pro-inflammatory responses.

BURKHOLDERIA CEPACIA: is an opportunistic pathogen that infects patients with debilitating underlying diseases. This study investigated the production of outer membrane vesicles (OMVs) by B. cepacia cultured with sub-minimum inhibitory concentrations (MICs) of antibiotics and examined their pathogenic roles both in vitro and in vivo. B. cepacia ATCC 25416 produced more OMVs under antibiotic stress conditions than controls. OMVs isolated from B. cepacia cultured in Luria-Bertani (LB) broth (OMVs/LB) induced cytotoxicity and the expression of pro-inflammatory cytokine genes in A549 cells in a dose-dependent manner. Host cell cytotoxicity and pro-inflammatory responses were significantly higher in A549 cells treated with B. cepacia OMVs cultured with 1/4 MIC of ceftazidime (OMVs/CAZ) than in the cells treated with OMVs/LB, OMVs cultured with 1/4 MIC of trimethoprim/sulfamethoxazole (OMVs/SXT), or OMVs cultured with 1/4 MIC of meropenem. Intratracheal injection of B. cepacia OMVs also induced histopathology in vivo in mouse lungs. Expressions of IL-1ß and TNF-α genes were significantly up-regulatedin the lungs of mice treated with OMVs/CAZ compared to mice administered other OMVs; the expression of the GRO-α gene, however, was significantly up-regulated in OMVs/SXT. In conclusion, OMVs produced by B. cepacia under different antibiotic stress conditions induce different host responses that may contribute to the pathogenesis of B. cepacia.

Genomic and phenotypic characterization of Burkholderia isolates from the potable water system of the International Space Station.

The opportunistic pathogens Burkholderia cepacia and Burkholderia contaminans, both genomovars of the Burkholderia cepacia complex (BCC), are frequently cultured from the potable water dispenser (PWD) of the International Space Station (ISS). Here, we sequenced the genomes and conducted phenotypic assays to characterize these Burkholderia isolates. All recovered isolates of the two species fall within monophyletic clades based on phylogenomic trees of conserved single-copy core genes. Within species, the ISS-derived isolates all demonstrate greater than 99% average nucleotide identity (with 95-99% of genomes aligning) and share around 90% of the identified gene clusters from a pangenomic analysis-suggesting that the two groups are each composed of highly similar genomic lineages and their members may have all stemmed from the same two founding populations. The differences that can be observed between the recovered isolates at the pangenomic level are primarily located within putative plasmids. Phenotypically, macrophage intracellularization and lysis occurred at generally similar rates between all ISS-derived isolates, as well as with their respective type-terrestrial strain references. All ISS-derived isolates exhibited antibiotic sensitivity similar to that of the terrestrial reference strains, and minimal differences between isolates were observed. With a few exceptions, biofilm formation rates were generally consistent across each species. And lastly, though isolation date does not necessarily provide any insight into how long a given isolate had been aboard the ISS, none of the assayed physiology correlated with either date of isolation or distances based on nucleotide variation. Overall, we find that while the populations of Burkholderia present in the ISS PWS each maintain virulence, they are likely are not more virulent than those that might be encountered on planet and remain susceptible to clinically used antibiotics.

Intracellular survival and innate immune evasion of Burkholderia cepacia: Improved understanding of quorum sensing-controlled virulence factors, biofilm, and inhibitors.

Burkholderia cepacia complex (Bcc) are opportunistic pathogens implicated with nosocomial infections, and high rates of morbidity and mortality, especially in individuals with cystic fibrosis (CF). B. cepacia are naturally resistant to different classes of antibiotics, and can subvert the host innate immune responses by producing quorum sensing (QS) controlled virulence factors and biofilms. It still remains a conundrum as to how exactly the bacterium survives the intracellular environment within the host cells of CF patients and immunocompromised individuals although the bacterium can invade human lung epithelial cells, neutrophils, and murine macrophages. The mechanisms associated with intracellular survival in the airway epithelial cells and the role of QS and virulence factors in B. cepacia infections in cystic fibrosis remain largely unclear. The current review focuses on understanding the role of QS-controlled virulence factors and biofilms, and provides additional impetus to understanding the potentials of QS-inhibitory strategies against B. cepacia.

Synergy between Active Efflux and Outer Membrane Diffusion Defines Rules of Antibiotic Permeation into Gram-Negative Bacteria.

Gram-negative bacteria are notoriously resistant to antibiotics, but the extent of the resistance varies broadly between species. We report that in significant human pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Burkholderia spp., the differences in antibiotic resistance are largely defined by their penetration into the cell. For all tested antibiotics, the intracellular penetration was determined by a synergistic relationship between active efflux and the permeability barrier. We found that the outer membrane (OM) and efflux pumps select compounds on the basis of distinct properties and together universally protect bacteria from structurally diverse antibiotics. On the basis of their interactions with the permeability barriers, antibiotics can be divided into four clusters that occupy defined physicochemical spaces. Our results suggest that rules of intracellular penetration are intrinsic to these clusters. The identified specificities in the permeability barriers should help in the designing of successful therapeutic strategies against antibiotic-resistant pathogens.IMPORTANCE Multidrug-resistant strains of Gram-negative pathogens rapidly spread in clinics. Significant efforts worldwide are currently directed to finding the rules of permeation of antibiotics across two membrane envelopes of these bacteria. This study created the tools for analysis of and identified the major differences in antibacterial activities that distinguish the permeability barriers of P. aeruginosa, A. baumannii, Burkholderia thailandensis, and B. cepacia We conclude that synergy between active efflux and the outer membrane barrier universally protects Gram-negative bacteria from antibiotics. We also found that the diversity of antibiotics affected by active efflux and outer membrane barriers is broader than previously thought and that antibiotics cluster according to specific biological determinants such as the requirement of specific porins in the OM, targeting of the OM, or specific recognition by efflux pumps. No universal rules of antibiotic permeation into Gram-negative bacteria apparently exist. Our results suggest that antibiotic clusters are defined by specific rules of permeation and that further studies could lead to their discovery.

Tuberculosis in an autosomal recessive case of chronic granulomatous disease due to mutation of the NCF1 gene

No disponible

Investigating the Role of the Host Multidrug Resistance Associated Protein Transporter Family in Burkholderia cepacia Complex Pathogenicity Using a Caenorhabditis elegans Infection Model.

This study investigated the relationship between host efflux system of the non-vertebrate nematode Caenorhabditis elegans and Burkholderia cepacia complex (Bcc) strain virulence. This is the first comprehensive effort to profile host-transporters within the context of Bcc infection. With this aim, two different toxicity tests were performed: a slow killing assay that monitors mortality of the host by intestinal colonization and a fast killing assay that assesses production of toxins. A Virulence Ranking scheme was defined, that expressed the toxicity of the Bcc panel members, based on the percentage of surviving worms. According to this ranking the 18 Bcc strains were divided in 4 distinct groups. Only the Cystic Fibrosis isolated strains possessed profound nematode killing ability to accumulate in worms' intestines. For the transporter analysis a complete set of isogenic nematode single Multidrug Resistance associated Protein (MRP) efflux mutants and a number of efflux inhibitors were interrogated in the host toxicity assays. The Bcc pathogenicity profile of the 7 isogenic C. elegans MRP knock-out strains functionality was classified in two distinct groups. Disabling host transporters enhanced nematode mortality more than 50% in 5 out of 7 mutants when compared to wild type. In particular mrp-2 was the most susceptible phenotype with increased mortality for 13 out 18 Bcc strains, whereas mrp-3 and mrp-4 knock-outs had lower mortality rates, suggesting a different role in toxin-substrate recognition. The use of MRP efflux inhibitors in the assays resulted in substantially increased (>40% on average) mortality of wild-type worms.

Investigating the role of matrix components in protection of Burkholderia cepacia complex biofilms against tobramycin.

BACKGROUND: Burkholderia cepacia complex (Bcc) organisms produce a wide variety of potential virulence factors, including exopolysaccharides (EPS), and exhibit intrinsic resistance towards many antibiotics. In the present study we investigated the contribution of Bcc biofilm matrix components, including extracellular DNA, cepacian and poly-ß-1,6-N-acetylglucosamine, to tobramycin susceptibility. METHODS: The in vitro bactericidal activity of tobramycin in combination with recombinant human DNase (rhDNase), NaClO and dispersin B was tested against Bcc biofilms. RESULTS: EPS degradation by NaClO pretreatment and specific PNAG degradation by dispersin B significantly increased the bactericidal effect of tobramycin towards some of the Bcc biofilms tested, including the strains of Burkholderia cenocepacia, B. cepacia and Burkholderia metallica. The presence of rhDNase during biofilm treatment and/or development had no influence on tobramycin activity. CONCLUSION: These results suggest that EPS play a role in tobramycin susceptibility of Bcc biofilms and that matrix degrading combination therapy could improve treatment of Bcc biofilm infections.

Proteomic profiling of Burkholderia cenocepacia clonal isolates with different virulence potential retrieved from a cystic fibrosis patient during chronic lung infection.

Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient's death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.

Burkholderia cepacia complex infection in an Adult Cystic Fibrosis unit in Madrid

Introduction: Burkholderia cepacia complex have emerged as significant pathogens in cystic fibrosis (CF) patients due to the risk of cepacia syndrome and the innate multi-resistance of the microorganisms to antibiotics. The aim of this study was to describe the antimicrobial susceptibility profiles, the genotypes and subtypes of BCC, and the clinical evolution of CF patients with BCC. Methods The lung function and Brasfield and Shwachman score were assessed in 12 patients. BCC were identified and susceptibility was studied by MicroScan (Siemens). Species and genospecies of BCC were confirmed by molecular methods in a Reference Centre (Majadahonda).Result sBCC were identified in 12 of 70 patients (17.1%) over a ten year period. The mean age to colonization by BCC was 24.4 years (SD: 7.71). B. cenocepacia was isolated in 4 patients (33.3%), B. contaminans was isolated in 3 patients (25%), both B. vietnamiensis and B. stabilis were isolated in 2 patients (16.7%), and B. cepacia, B. multivorans and B. late were isolated in one patient (8.3%). Among the B. cenocepacia, subtype IIIa was identified in two strains, and subtype IIIb was identified in the other two strains. There was susceptibility to meropenem in 90% of BCC, 80% to cotrimoxazole, 60% to minocycline, 50% to ceftazidime, and 40% to levofloxacin. Conclusions B. cenocepacia was the most prevalent species among the BCC isolated in CF adult patients, and subtypes IIIa and IIIb were identified in the 50% of the strains. Meropenem and cotrimoxazole showed the best activity

Introduction: Burkholderia cepacia complex have emerged as significant pathogens in cystic fibrosis (CF)patients due to the risk of cepacia syndrome and the innate multi-resistance of the microorganisms to antibiotics. The aim of this study was to describe the antimicrobial susceptibility profiles, the genotypes and subtypes of BCC, and the clinical evolution of CF patients with BCC. Methods: The lung function and Brasfield and Shwachman score were assessed in 12 patients.BCC were identified and susceptibility was studied by MicroScan (Siemens). Species and genospecies of BCC were confirmed by molecular methods in a Reference Centre (Majadahonda).Results: BCC were identified in 12 of 70 patients (17.1%) over a ten year period. The mean age to colonization by BCC was 24.4 years (SD: 7.71). B. cenocepacia was isolated in 4 patients (33.3%), B. contaminans was isolated in 3 patients (25%), both B. vietnamiensis and B. stabilis were isolated in 2 patients (16.7%), and B. cepacia, B. multivorans and B. late were isolated in one patient (8.3%). Among the B. cenocepacia, subtype IIIa was identified in two strains, and subtype IIIb was identified in the other two strains. There was susceptibility to meropenem in 90% of BCC, 80% to cotrimoxazole, 60% to minocycline, 50% to ceftazidime, and 40% to levofloxacin. Conclusions: B. cenocepacia was the most prevalent species among the BCC isolated in CF adult patients, and subtypes IIIa and IIIb were identified in the 50% of the strains. Meropenem and cotrimoxazole showed the best activity

Comparative transcriptomic analysis of the Burkholderia cepacia tyrosine kinase bceF mutant reveals a role in tolerance to stress, biofilm formation, and virulence.

The bacterial tyrosine-kinase (BY-kinase) family comprises the major group of bacterial enzymes endowed with tyrosine kinase activity. We previously showed that the BceF protein from Burkholderia cepacia IST408 belongs to this BY-kinase family and is involved in the biosynthesis of the exopolysaccharide cepacian. However, little is known about the extent of regulation of this protein kinase activity. In order to examine this regulation, we performed a comparative transcriptome profile between the bceF mutant and wild-type B. cepacia IST408. The analyses led to identification of 630 genes whose expression was significantly changed. Genes with decreased expression in the bceF mutant were related to stress response, motility, cell adhesion, and carbon and energy metabolism. Genes with increased expression were related to intracellular signaling and lipid metabolism. Mutation of bceF led to reduced survival under heat shock and UV light exposure, reduced swimming motility, and alteration in biofilm architecture when grown in vitro. Consistent with some of these phenotypes, the bceF mutant demonstrated elevated levels of cyclic-di-GMP. Furthermore, BceF contributed to the virulence of B. cepacia for larvae of the Greater wax moth, Galleria mellonella. Taken together, BceF appears to play a considerable role in many cellular processes, including biofilm formation and virulence. As homologues of BceF occur in a number of pathogenic and plant-associated Burkholderia strains, the modulation of bacterial behavior through tyrosine kinase activity is most likely a widely occurring phenomenon.

[The express diagnostic of microorganisms affecting respiratory tract of patients with mucoviscidosis].

The shared bacteria Burkholderia capacia complex and Achromobacter sp. infect the respiratory tract of patients with mucoviscidosis brining on disorders of respiratory patency. Burkholderia capacia complex is characterized by transmissivity and higher lethality of patients infected by Burkholderia. Hence, the importance of differentiation of these phenotypically similar microorganisms is obvious. The developed express technique of diagnostic includes the separation of DNA from phlegm amplification and sequenation was fragments of genes recA, gltB, gyrB, 16S rDNA. The evaluation of products of amplification of genes recA, gltB makes it possible to differentiate Burkholderia capacia complex and Achromobacter sp. The analysis of successions of recA, gltB, gyrB makes it possible to identify genotype of Burkholderia capacia complex on the basis of data of allele profiles of strains of Burkholderia capacia complex circulating in Russia. The succession of gene 16S rDNA makes it possible to determine the taxonomic position of microorganism dominating in phlegm and not belonging to Burkholderia capacia complex or Achromobacter sp. The real time polymerase chain reaction in presence of intercalating dye Sybr Green I, DMSO and D(+)-trehalose makes it possible to differentiate Burkholderia capacia complex from other microorganisms infecting respiratory tract of patients with mucoviscidosis. This approach provides additional reduction of diagnostic duration and decrease possibility of contamination.

[Burkholderia cepacia persistence in patients with mucoviscidosis].

AIM: Study features of persistence of Burkholderia cepacia in mucoviscidosis patients. MATERIALS AND METHODS: In the period from 2008 to 2009, 56 B. cepacia strains isolated from children with mucoviscidosis were obtained. 114 medical histories of children with mucoviscidosis from various age groups were analyzed. The developed algorithm for identification and typing including phenotype and molecular biology methods was used to identify B. cepacia bacteria. Strain genotyping was carried out by RAPD-PCR with random oligonucleotide primer as well as pulse-electrophoresis. RESULTS: Persistence of associations ofmicroogranisms in 59.4% of cases was established to be the feature of persistent infection in mucoviscidosis. The feature of persistence of B. cepacia strains in patients with diagnosis ofmuco-viscidosis mixed form, severe course is persistence in association with Pseudomonas aeruginosa. B. cepacia bacteria that can persist in mucoviscidosis patients are characterized by resistance to many antibiotics. A prolonged (up to 1 year and 5 months) persistence of B. cepacia strains isolated from 1 patient was proven by using microflora monitoring of lower respiratory tract. CONCLUSION: B. cepacia bacteria may colonize lower respiratory tract of mucoviscidosis patients, persist for a long time and be transmitted between patients.

Bacteriemias por Burkholderia cepacia: análisis prospectivo de 33 episodios / Burkholderia cepacia bacteremia: a prospective analysis of 33 episodes

Introducción: El objetivo de este estudio es la descripción de las características clínicas y evolución de los episodios de bacteriemia por Burkholderia cepacia, conocer la sensibilidad antibiótica de las cepas aisladas y analizar las diferencias entre los casos procedentes de brotes epidémicos y los episodios esporádicos. Material y métodos: Se recogieron de forma prospectiva desde 1993 a 2009 todas las bacteriemias por B. cepacia en un hospital universitario. Resultados: Se incluyeron 33 episodios, de los cuales 21 se concentraron en dos brotes (9 en 1994 y 12 en 2006). Los casos pertenecientes a los brotes tuvieron una mediana de edad de 58 años, el 45 % tenía alguna neoplasia, la mediana de días desde el ingreso hasta la bacteriemia fue de 15 (rango 0- 120) y el 82% había recibido algún antibiótico. Los focos más frecuentes fueron catéter (48%) y desconocido (33%). Por otro lado, los casos esporádicos llevaban más días ingresados (una mediana de 25 días frente a 11, p=0,041) y mostraron mayor tendencia a presentar neoplasia (83% frente a 33%, p=0,083). El patrón de resistencias antibióticas fue muy variado y el único antibiótico activo frente a todos los aislados fue cotrimoxazol. Conclusiones: B. cepacia es un microrganismo infrecuente, que afecta a pacientes con ingresos prolongados y comorbilidad importante. La identificación de más de un caso en un corto periodo de tiempo obliga a descartar un posible brote(AU)

Introduction: The aim of this study is to describe clinical characteristics and outcome of Burkholderia cepacia bacteraemia, susceptibility of the isolates and differences between cases from epidemic outbreaks and sporadic cases. Material and methods: From 1993 to 2009, episodes of B. cepacia bacteraemia were prospectively collected in a university hospital. Results: A total of 33 episodes were included, of which 21 were part of two outbreaks (9 in 1994 and 12 in 2006). Outbreak cases had a median age of 58 years, 45% had neoplasia, median length of stay until bacteraemia was 15 d (range 0- 120) and 82% had received an antibiotic. The most prevalent sources of bacteraemia were catheter (48%) and unknown (33%). On the other hand, sporadic cases stayed longer until diagnosis (median 25 days versus 11, p=0.041) and showed a trend to have neoplasia more frequently (83% versus 33%, p=0.083). Susceptibility to antibiotics was varied and co-trimoxazole was the only active agent against all strains. Conclusions: B. cepacia is an uncommon pathogen, which affects patients with prolonged hospitalization and severe comorbidities. The identification of more than one case in a short term of time should raise the suspicion of an outbreak(AU)

Colonización por Pandoraea sputorum en un paciente con fibrosis quística / Pandoraea Sputorum Colonization in a Patient with Cystic Fibrosis

La fibrosis quística (FQ) es una enfermedad producida por un defecto de la proteína reguladora de la conductancia transmembrana CFTR. En la actualidad la morbimortalidad asociada a FQ está relacionada fundamentalmente con la afectación pulmonar consecuencia de este defecto. Con el progreso de la enfermedad se incrementa el aislamiento de bacilos gramnegativos no fermentadores como colonizadores en estos pacientes.El género Pandoraea surge de una reclasificación de especies integradas en el «complejo Burkholderia cepacia», y comprende 9 especies sensibles solamente a tetraciclinas, imipenem y cotrimoxazol.Describimos el primer caso clínico, en España, de colonización por Pandoraea sputorum en una paciente diagnosticada de FQ a los 11 años que, tras varias colonizaciones previas por diferentes especies de Pseudomonas, en septiembre de 2005 se aisló en esputo un bacilo gramnegativo identificado por secuenciación y espectrometría de masas (MALDITOF-MS) como P. sputorum, resultando solo sensible a piperacilina-tazobactam, cotrimoxazol e imipenem. Desde 2005 a 2008 se demostró la colonización crónica por este microorganismo, asociándose a un deterioro de la función pulmonar que se recuperó tras tratamiento con piperacilina-tazobactam e imipenem. En 2010 se volvió a aislar este microorganismo y se trató con imipenem, fármaco al que la paciente respondió favorablemente.En la actualidad se desconoce si este microorganismo es un colonizador crónico, produce una infección transitoria o constituye un problema importante en el paciente con FQ, pero dadas las características especiales de sensibilidad a antimicrobianos, la identificación correcta de este género es fundamental. La espectrometría de masas parece ser una técnica válida y más rápida que los métodos de secuenciación para la identificación de estas especies(AU)

Cystic fibrosis (CF) is a disease produced by a defect in the transmembrane conductance regulator protein, CFTR. Currently, the morbidity and mortality associated with CF are fundamentally related with the lung affectation that is a consequence of this defect. With the progression of the disease, there is an increase in the isolation of non-fermenting gram-negative bacilli colonizing these patients.The genus Pandoraea arises from a reclassification of species included within the “Burkholderia cepacia complex”. It is made up of 9 species susceptible only to tetracycline, imipenem and cotrimoxazole.We report the first clinical case in Spain of colonization by Pandoraea sputorum in a patient diagnosed with CF at the age of eleven. After several previous colonizations by different Pseudomonas species in September 2005, a gram-negative bacillus was isolated in sputum, which was identified by sequencing and mass spectrometry (MALDITOF-MS) as P. sputorum, only sensitive to piperacillin-tazobactam, cotrimoxazole and imipenem. From 2005 to 2008, chronic colonization by this microorganism was associated with deterioration in lung function that was recuperated after treatment with piperacillin-tazobactam and imipenem. In 2010, this microorganism was once again isolated and treated with imipenem, to which the patient responded favorably.Currently, it is not known whether this microorganism is a chronic colonizer, whether it produces a transitory infection or whether it constitutes an important problem in CF patients, but given its special characteristics of sensitivity to anti-microbial drugs, the correct identification of this genus is essential. Mass spectrometry seems to be a valid technique that is faster than sequencing methods for identifying these species(AU)

Inactivation of Burkholderia cepacia complex phage KS9 gp41 identifies the phage repressor and generates lytic virions.

The Burkholderia cepacia complex (BCC) is made up of at least 17 species of gram-negative opportunistic bacterial pathogens that cause fatal infections in patients with cystic fibrosis and chronic granulomatous disease. KS9 (vB_BcenS_KS9), one of a number of temperate phages isolated from BCC species, is a prophage of Burkholderia pyrrocinia LMG 21824. Transmission electron micrographs indicate that KS9 belongs to the family Siphoviridae and exhibits the B1 morphotype. The 39,896-bp KS9 genome, comprised of 50 predicted genes, integrates into the 3' end of the LMG 21824 GTP cyclohydrolase II open reading frame. The KS9 genome is most similar to uncharacterized prophage elements in the genome of B. cenocepacia PC184 (vB_BcenZ_ PC184), as well as Burkholderia thailandensis phage phiE125 and Burkholderia pseudomallei phage phi1026b. Using molecular techniques, we have disrupted KS9 gene 41, which exhibits similarity to genes encoding phage repressors, producing a lytic mutant named KS9c. This phage is incapable of stable lysogeny in either LMG 21824 or B. cenocepacia strain K56-2 and rescues a Galleria mellonella infection model from experimental B. cenocepacia K56-2 infections at relatively low multiplicities of infection. These results readily demonstrate that temperate phages can be genetically engineered to lytic form and that these modified phages can be used to treat bacterial infections in vivo.

Susceptibility of Caenorhabditis elegans to Burkholderia infection depends on prior diet and secreted bacterial attractants.

The nematode Caenorhabditis elegans may be killed by certain pathogenic bacteria and thus is a model organism for studying interactions between bacteria and animal hosts. However, growing nematodes on prey bacteria may influence their susceptibility to potential pathogens. A method of axenic nematode culture was developed to isolate and quantify interactions between C. elegans and potentially pathogenic strains of the Burkholderia cepacia complex. Studying these dynamics in liquid solution rather than on agar surfaces minimized nematode avoidance behavior and resolved more differences among isolates. Most isolates of B. cenocepacia, B. ambifaria and B. cepacia caused 60-80% mortality of nematodes after 7 days, whereas isolates of B. multivorans caused less mortality (<25%) and supported nematode reproduction. However, some B. cenocepacia isolates recovered from chronic infections were much less virulent (5-28% mortality). As predicted, prior diet altered the outcome of interactions between nematodes and bacteria. When given the choice between Burkholderia and E. coli as prey on agar, axenically raised nematodes initially preferred most lethal Burkholderia isolates to E. coli as a food source, but this was not the case for nematodes fed E. coli, which avoided toxic Burkholderia. This food preference was associated with the cell-free supernatant and thus secreted compounds likely mediated bacterial-nematode interactions. This model, which isolates interactions between bacteria and nematodes from the effects of prior feeding, demonstrates that bacteria can influence nematode behavior and their susceptibility to pathogens.

[Persistence of Burkholderia cenocepacia bacteria in vivo in dependence of their ability to form biofilms].

AIM: To select the most susceptible line of mice which allows to conduct comparative studies of infectious process caused by different strains of B. cepacia in order to explore correlation between ability to form biofilms and persistence of bacteria in organs of infected animals. MATERIALS AND METHODS: Strain B. cenocepacia 370, which is a clinical isolate, and its mutants with modified ability to form biofilms were used. Conditional microbiologic methods and biological models of intraperitoneal and intranasal inoculation of mice belonging to 4 lines: BALB/c, BLACK, I/St, and A/Sn derived in Central Institute of Tuberculosis were employed. Criteria of persistence was duration of isolation of different strains of bacteria from lungs and spleen of inoculated animals as well as number of CFU. RESULTS: The most susceptible line of mice which enables to conduct comparative studies of infectious process caused by Burkholderia species was determined. It was shown that even after intraperitoneal inoculation the agent was better preserved in lungs than in spleen that corresponds to natural localization of this infection. At any time of observation the number of cells of mutant strain, which is a superproducer of biofilms, isolated from organs of inoculated mice was 2 - 10 times higher than number of isolated cells of mutant, which do not produce biofilms. CONCLUSION: Correlation of more prolonged persistence of B. cenocepacia in organs of inoculated animals in vivo with ability of the agent to form biofilms determined in vitro is experimentally established. The susceptible line of mice which allows to conduct comparative studies of dynamics of infectious process caused by various strains of Burkholderia species was revealed. It was shown that irrespective from method of inoculation B. cepacia are able to continuously persist in organism of susceptible animals with lungs as a predominant localization.

Respiratory microbiology of patients with cystic fibrosis in the United States, 1995 to 2005.

BACKGROUND: Numerous improvements in diagnostic and therapeutic strategies for patients with cystic fibrosis (CF) have occurred during the past 2 decades. We hypothesized that these changes could impact trends in respiratory microbiology. METHODS: Data from the Cystic Fibrosis Foundation Patient Registry were used to examine trends in the incidence and prevalence of bacterial pathogens isolated from patients with CF in the United States from 1995 to 2005. RESULTS: The number of patients with CF in the patient registry increased from 19,735 in 1995 to 23,347 in 2005. During the study period, the reported annual prevalence of Pseudomonas aeruginosa significantly declined from 60.4% in 1995 to 56.1% in 2005 (p < 0.001). The decline was most marked in children 6 to 10 years old (48.2 to 36.1%) and adolescents 11 to 17 years old (68.9 to 55.5%). Both the incidence (21.7% in 1995 and 33.2% in 2005) and prevalence (37.0% in 1995 and 52.4% in 2005) of methicillin-susceptible Staphylococcus aureus significantly increased and the age-specific prevalence was highest in patients 6 to 17 years old. The prevalence of methicillin-resistant S aureus increased from 0.1% in 1995 to 17.2% in 2005 and from 2002 to 2005 was highest in adolescents 11 to 17 years old. Both the prevalence and incidence of Burkholderia cepacia complex declined, while the prevalence of Haemophilus influenzae, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans increased. CONCLUSIONS: Data from the patient registry suggest that the epidemiology of bacterial pathogens in patients with CF changed during the study period. Future studies should continue to monitor changing trends and define the association between these trends and care practices in CF.