RESUMEN

Burkholderia cepacia complex (Bcc) species are opportunistic pathogens widely distributed in the environment and often infect people with cystic fibrosis (CF). This study aims to determine which genomovars of the Bcc can cause infections in non-CF patients from a tertiary care hospital in Mexico and if they carry virulence factors that could increase their pathogenicity. We identified 23 clinical isolates that carry the recA gene. Twenty-two of them belongs to the genomovar V (B. vietnamiensis) and one to the genomovar II (B. multivorans). Thirteen pulsotypes were identified among 22 B. vietnamiensis isolates. All clinical isolates produced biofilm were motile and cytotoxic on murine macrophage-like RAW264.7 and in A549 human lung epithelial cells. In conclusion, B. vietnamiensis causes infections in non-CF patients in a tertiary care hospital in Mexico, rapid identification of this pathogen can help physicians to establish a better antimicrobial treatment.

Asunto(s)

Infecciones por Burkholderia , Complejo Burkholderia cepacia , Burkholderia cepacia , Fibrosis Quística , Humanos , Animales , Ratones , Burkholderia cepacia/genética , Infecciones por Burkholderia/epidemiología , México/epidemiología , Centros de Atención Terciaria , Reacción en Cadena de la Polimerasa , Complejo Burkholderia cepacia/genética , Fibrosis Quística/complicaciones

RESUMEN

Patients undergoing hemodialysis are at an increased risk for bloodstream infections (BSIs). Infection usually occurs because of contamination of water supply, water treatment, distribution systems, or reprocessing dialyzers. Here, we report an outbreak of BSIs caused by Stenotrophomonas maltophilia (n = 21) and Burkholderia cepacia (n = 22) among dialyzed patients at a large hemodialysis center in Brazil. Overall, three patients died (7%), two of which had bacteremia caused by S. maltophilia and the other had a B. cepacia infection. We collected water samples from different points of the hemodialysis system for culture and typing. Genetic patterns were identified through polymerase chain reaction-random amplified polymorphic DNA (PCR-RAPD) and pulsed-field gel electrophoresis. The same genotypes of S. maltophilia and B. cepacia recovered from blood cultures were found in dialysis water. Also, multiple genetic profiles were identified among water isolates, suggesting heavy contamination. Bacteremia cases persisted even after implementing standard control measures, which led us to believe that the piping system was contaminated with microbial biofilms. Soon after we changed the entire plumbing system, reported cases dropped back to the number typically expected, and the outbreak came to an end.

Asunto(s)

Infecciones por Burkholderia/epidemiología , Burkholderia cepacia/aislamiento & purificación , Brotes de Enfermedades , Infecciones por Bacterias Gramnegativas/epidemiología , Diálisis Renal/efectos adversos , Stenotrophomonas maltophilia/aislamiento & purificación , Adulto , Anciano , Anciano de 80 o más Años , Brasil/epidemiología , Infecciones por Burkholderia/etiología , Infecciones por Burkholderia/prevención & control , Burkholderia cepacia/clasificación , Burkholderia cepacia/genética , Desinfección/métodos , Femenino , Infecciones por Bacterias Gramnegativas/etiología , Infecciones por Bacterias Gramnegativas/prevención & control , Humanos , Masculino , Persona de Mediana Edad , Diálisis Renal/normas , Sepsis/epidemiología , Sepsis/etiología , Sepsis/prevención & control , Stenotrophomonas maltophilia/clasificación , Stenotrophomonas maltophilia/genética

RESUMEN

Microbiota presumably plays an essential role in inhibiting pathogen colonization and in the maintenance of health in oysters, but limited data exist concerning their different growth phases and conditions. We analyzed the bacterial microbiota composition of two commercial oysters: Crassostrea gigas and Crassostrea corteziensis. Differences in microbiota were assayed in three growth phases: post-larvae at the hatchery, juvenile, and adult at two grow-out cultivation sites. Variations in the microbiota were assessed by PCR analysis of the 16S rRNA gene in DNA extracted from depurated oysters. Restriction fragment length polymorphism (RFLP) profiles were studied using Dice's similarity coefficient (Cs) and statistical principal component analysis (PCA). The microbiota composition was determined by sequencing temperature gradient gel electrophoresis (TGGE) bands. The RFLP analysis of post-larvae revealed homology in the microbiota of both oyster species (Cs > 88 %). Dice and PCA analyses of C. corteziensis but not C. gigas showed differences in the microbiota according to the cultivation sites. The sequencing analysis revealed low bacterial diversity (primarily ß-Proteobacteria, Firmicutes, and Spirochaetes), with Burkholderia cepacia being the most abundant bacteria in both oyster species. This study provides the first description of the microbiota in C. corteziensis, which was shown to be influenced by cultivation site conditions. During early growth, we observed that B. cepacia colonized and remained strongly associated with the two oysters, probably in a symbiotic host-bacteria relationship. This association was maintained in the three growth phases and was not altered by environmental conditions or the management of the oysters at the grow-out site.

Asunto(s)

Bacterias/genética , Crassostrea/crecimiento & desarrollo , Crassostrea/microbiología , Metagenoma , ARN Ribosómico 16S/genética , Animales , Acuicultura , Bacterias/clasificación , Bacterias/aislamiento & purificación , Burkholderia cepacia/clasificación , Burkholderia cepacia/genética , Burkholderia cepacia/aislamiento & purificación , ADN Bacteriano/análisis , ADN Bacteriano/genética , Electroforesis en Gel de Gradiente Desnaturalizante , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN

RESUMEN

Polyhydroxyalkanoates (PHA) are natural polyesters stored by a wide range of bacteria as carbon source reserve. Due to its chemical characteristics and biodegradability PHA can be used in chemical, medical and pharmaceutical industry for many human purposes. Over the past years, few Burkholderia species have become known for production of PHA. Aside from that, these bacteria seem to be interesting for discovering new PHA compositions which is important to different industrial applications. In this paper, we introduce two new strains which belong either to Burkholderia cepacia complex (Bcc) or genomovar-type, Burkholderia cepacia SA3J and Burkholderia contaminans I29B, both PHA producers from unrelated carbon sources. The classification was based on 16S rDNA and recA partial sequence genes and cell wall fatty acids composition. These two strains were capable to produce different types of PHA monomers or precursors. Unrelated carbon sources were used for growth and PHA accumulation. The amount of carbon source evaluated, or mixtures of them, was increased with every new experiment until it reaches eighteen carbon sources. As first bioprospection experiments staining methods were used with colony fluorescent dye Nile Red and the cell fluorescent dye Nile Blue A. Gas chromatography analysis coupled to mass spectrometry was used to evaluate the PHA composition on each strain cultivated on different carbon sources. The synthesized polymers were composed by short chain length-PHA (scl-PHA), especially polyhydroxybutyrate, and medium chain length-PHA (mcl-PHA) depending on the carbon source used.

Asunto(s)

Secuencia de Bases , Burkholderia cepacia/genética , Carbono/análisis , Espectrometría de Masas , Polihidroxialcanoatos/análisis , Polímeros/análisis , Cromatografía de Gases , Microbiología Industrial , Métodos , Métodos

RESUMEN

Identification of all important community members as well as of the numerically dominant members of a community are key aspects of microbial community analysis of bioreactor samples. A systematic study was conducted with artificial consortia to test whether denaturing gradient gel electrophoresis (DGGE) is a reliable technique to obtain such community data under conditions where results would not be affected by differences in DNA extraction efficiency from cells. A total of 27 consortia were established by mixing DNA extracted from Escherichia coli K12, Burkholderia cepacia and Stenotrophomonas maltophilia in different proportions. Concentrations of DNA of single organisms in the consortia were either 0.04, 0.4 or 4ng/microl. DGGE-PCR of genomic DNA with primer sets targeted at the V3 and V6-V8 regions of the 16S rDNA failed to detect the three community members in only 7% of consortia, but provided incorrect information about dominance or co-dominance for 85% and 89% of consortia with the primer sets for the V6-V8 and V3 regions, respectively. The high failure rate in detection of dominant B. cepacia with the primers for the V6-V8 region was attributable to a single nucleotide primer mismatch in the target sequences of both, the forward and reverse primer. Amplification bias in PCR of E. coli and S. maltophilia for the V6-V8 region and for all three organisms for the V3 region occurred due to interference of genomic DNA in PCR-DGGE, since a nested PCR approach, where PCR-DGGE was started from mixtures of 16S rRNA genes of the organisms, provided correct information about the relative abundance of original DNA in the sample. Multiple bands were not observed in pure culture amplicons produced with the V6-V8 primer pair, but pure culture V3 DGGE profiles of E. coli, S. maltophilia and B. cepacia contained 5, 3 and 3 bands, respectively. These results demonstrate DGGE was suitable for identification of all important community members in the three-membered artificial consortium, but not for identification of the dominant organisms in this small community. Multiple DGGE bands obtained for single organisms with the V3 primer pair could greatly confound interpretation of DGGE profiles.

Asunto(s)

Reactores Biológicos , Burkholderia cepacia/genética , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Electroforesis/métodos , Escherichia coli/genética , Gammaproteobacteria/genética , Burkholderia cepacia/aislamiento & purificación , Cartilla de ADN , Escherichia coli/aislamiento & purificación , Gammaproteobacteria/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Microbiología del Agua

RESUMEN

A mineral phosphate solubilizing bacterium, Burkholderia cepacia DA23 has been isolated from cultivated soils. Phosphate-solubilizing activities of the strain against three types of insoluble phosphate were quantitatively determined. When 3 percent of glucose concentration was used for carbon source, the strain had a marked mineral phosphate-solubilizing activity. Mineral phosphate solubilization was directly related to the pH drop by the strain. Analysis of the culture medium by high pressure liquid chromatography identified gluconic acid as the main organic acid released by Burkholderia cepacia DA23. Gluconic acid production was apparently the result of the glucose dehydrogenase activity and glucose dehydrogenase was affected by phosphate regulation.

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Uma bactéria capaz de solubilizar fosfato mineral, Burkholderia cepacea DA23, foi isolada de solo cultivado. A capacidade dessa bactéria solubilizar o fosfato de três tipos de fosfato insolúvel foi quantificada. Quando foi utilizada glicose a 3 por cento como fonte de carbono, a bactéria apresentou uma intensa atividade solubilizante de fosfato, sendo a solubilização diretamente relacionada com a queda de pH causada pela bactéria. A análise do meio de cultura por cromatografia líquida de alta pressão indicou o ácido glicônico como principal ácido produzido por Burkholderia cepacea DA23. Aparentemente, a produção de ácido glicônico foi causada pela atividade da glicose desidrogenase. A enzima foi afetada pela regulação do fosfato.

Asunto(s)

Burkholderia cepacia/genética , Burkholderia cepacia/aislamiento & purificación , Medios de Cultivo , Fosfatos/análisis , Glucosa/análisis , Técnicas In Vitro , Suelo , Cromatografía Líquida de Alta Presión , Métodos , Solubilidad , Virulencia

RESUMEN

Knowledge about the virulence mechanisms of species from the Burkholderia cepacia complex (BCC) is still limited. The genomovar heterogeneity and production of different virulence factors are likely to contribute to the variation in the clinical outcome observed in BCC-infected cystic fibrosis (CF) patients. Therefore, in this study we investigated the genetic polimorphism, the presence of genetic makers associated with virulence and transmissibility in BCC, and the profile of exoenzyme production of 59 BCC isolates obtained from 59 CF patients attending the reference CF centre in Rio de Janeiro, Brazil. The DNA sequence analyses of the recA gene allowed us to identify 40 of these 59 BCC species as being B. cenocepacia, 9 as B. vietnamiensis, 6 as B. multivorans and 4 as B. ambifaria. The assessment of the bacterial genetic polymorphism by PFGE revealed that B. cenocepacia and the B. multivorans isolates belonged to four and two different PFGE profiles with prevalence of two clones, A and B, respectively. All B. vietnamiensis and B. ambifaria belonged to only one PFGE profile (J and E, respectively). None of the isolates exhibited the genetic markers cblA and BCESM, assessed by polymerase chain reaction. In contrast, the profile of enzymatic activity, assessed by phenotypic methods, differed among the BCC species: protease activity was detected only in B. cenocepacia and B. ambifaria isolates, whereas only B. vietnamiensis isolates produced hemolysin. Although the phospholipase C activity was similar among the different species, the level of lipase activity produced by B. multivorans was higher than in the other species. We speculate that the differential characteristics of exoenzyme production may account for the differences in the pathogenic potentials of each BCC species.

Asunto(s)

Infecciones por Burkholderia/microbiología , Burkholderia cepacia/aislamiento & purificación , Burkholderia/aislamiento & purificación , Fibrosis Quística/microbiología , Enfermedades Pulmonares/microbiología , Proteínas Bacterianas/análisis , Técnicas de Tipificación Bacteriana , Biomarcadores/análisis , Brasil , Burkholderia/enzimología , Burkholderia/genética , Burkholderia cepacia/enzimología , Burkholderia cepacia/genética , Electroforesis en Gel de Campo Pulsado , Enzimas/análisis , Humanos , Polimorfismo Genético , Virulencia/genética

RESUMEN

A multiplex PCR method was developed to identify P. aeruginosa, B. cepacia complex, and S. maltophilia directly in sputum and oropharyngeal samples from CF patients. One hundred and six patients (53 male, and 53 female) attending our pulmonology clinic were studied from September 2000-April 2001. Two hundred and fifty-seven samples were cultured in selective media and submitted to multiplex PCR reactions, using three primer pairs targeting specific genomic sequences of each species, with an additional primer pair targeting a stretch of ribosomal 16S DNA, universal for bacteria, to act as a control. P. aeruginosa was isolated by culture in 56% of samples, B. cepacia complex in 4.3%, and S. maltophilia in 2.7%, while multiplex PCR identified P. aeruginosa in 78.7%, B. cepacia complex in 3.9%, and S. maltophilia in 3.1% of samples. Multiplex PCR results were verified by PCR reactions using different species-specific primers described in the literature and DNA sequencing of amplicons from a few samples. Comparing to culture results, the sensitivity and specificity values of multiplex PCR for bacterial identification were, respectively, 97.2% and 45.5% for P. aeruginosa, 45.5% and 97.9% for B. cepacia complex, and 40% and 97.6% for S. maltophilia. All 10 multiplex PCR-positive results for B. cepacia complex were confirmed using other species-specific primers described in the literature, while this approach confirmed results for S. maltophilia identification in 7/8 samples (87.5%). Sequencing of amplicons from samples culture-negative but multiplex PCR-positive for P. aeruginosa and B. cepacia complex confirmed their identity, while minor nucleotide differences among amplicons ruled out the hypothesis of PCR contamination.

Asunto(s)

Burkholderia cepacia/genética , Burkholderia cepacia/aislamiento & purificación , Fibrosis Quística/microbiología , Reacción en Cadena de la Polimerasa/métodos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/aislamiento & purificación , Adolescente , Adulto , Niño , Preescolar , Cartilla de ADN , ADN Bacteriano/análisis , Femenino , Humanos , Masculino , Orofaringe/microbiología , Sensibilidad y Especificidad , Esputo/microbiología

RESUMEN

PCR tests were used to assign genomovar status to 39 non-cystic fibrosis (non-CF) and 11 CF Burkholderia cepacia complex isolates from patients in hospitals in Recife, Brazil. Non-CF isolates were assigned to genomovar IIIA (71.8%), genomovar I (15.4%), B. vietnamiensis (7.7%), and B. multivorans (5.1%). CF isolates were assigned to genomovar IIIA (18.2%), B. vietnamiensis (18.2%), and genomovar I (9.1%). Six CF isolates sharing recA PCR-restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) patterns could not be assigned to a genomovar. 16S rDNA sequence obtained from these isolates indicated a closest relationship to B. anthina, but the recA sequence was equally divergent from several genomovars. PCR screening indicated the presence of cblA in only two isolates, whereas the B. cepacia epidemic strain marker was found in 22 of 28 genomovar IIIA isolates. A type III secretion gene was detected in all but genomovar I isolates. RAPD and PCR-RFLP assays, targeting both recA and fliC, indicated a large amount of genetic variability among the isolates, with many novel patterns being observed. Nine genomovar IIIA isolates from different non-CF patients and clinical sources had identical genotypes, indicating the presence of a common clone.

Asunto(s)

Técnicas de Tipificación Bacteriana/métodos , Burkholderia cepacia/clasificación , Burkholderia cepacia/genética , Brasil , Niño , Preescolar , Fibrosis Quística/microbiología , Flagelina/genética , Marcadores Genéticos , Humanos , Lactante , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Técnica del ADN Polimorfo Amplificado Aleatorio , Rec A Recombinasas/genética

RESUMEN

We report a polyclonal outbreak of bacteraemia involving 24 patients at a haemodialysis facility in Recife (Brazil). During the outbreak period (4 June to 11 July, 2001), three Burkholderia cepacia complex strains were isolated from human blood and from various water samples collected at different sites in the haemodialysis unit and from dialysate fluids. Out of 14 patients with positive blood cultures, six were infected by Burkholderia cepacia complex bacteria: three with Burkholderia cepacia genomovar III, two with a first strain of Burkholderia vietnamiensis, and one with the Burkholderia cepacia genomovar III strain and a second B. vietnamiensis strain.

Asunto(s)

Bacteriemia/microbiología , Infecciones por Burkholderia/microbiología , Burkholderia cepacia , Infección Hospitalaria/microbiología , Brotes de Enfermedades/estadística & datos numéricos , Diálisis Renal , Bacteriemia/epidemiología , Bacteriemia/prevención & control , Técnicas de Tipificación Bacteriana , Brasil/epidemiología , Infecciones por Burkholderia/epidemiología , Infecciones por Burkholderia/prevención & control , Burkholderia cepacia/clasificación , Burkholderia cepacia/genética , Recuento de Colonia Microbiana , Infección Hospitalaria/epidemiología , Infección Hospitalaria/prevención & control , ADN Bacteriano/análisis , ADN Bacteriano/genética , Brotes de Enfermedades/prevención & control , Electroforesis en Gel de Campo Pulsado , Soluciones para Hemodiálisis , Humanos , Control de Infecciones , Polimorfismo de Longitud del Fragmento de Restricción , Diálisis Renal/efectos adversos , Serotipificación , Microbiología del Agua , Abastecimiento de Agua

RESUMEN

The Burkholderia cepacia complex presently comprises nine genomovars: B. cepacia (genomovar I), B. multivorans (genomovar II), B. cepacia genomovar III, B. stabilis (genomovar IV), B. vietnamiensis (genomovar V), B. cepacia genomovar VI, B. ambifaria (genomovar VII), B. anthina (genomovar VIII) and B. pyrrocinia (genomovar IX). Strains of each genomovar can colonise the respiratory tract of cystic fibrosis (CF) patients. However, the majority of infections in CF patients are caused by B. multivorans and B. cepacia genomovar III isolates. Accurate genomovar-level identification is best achieved through a polyphasic approach combining phenotypic and genotypic analyses. In the present study, the sensitivity and specificity of recA-based genomovar specific primer pairs were evaluated with a collection of 508 B. cepacia complex isolates representing all nine genomovars. The assays for the identification of B. multivorans (sensitivity and specificity, 100%), B. cepacia genomovar III (sensitivity, 92%; specificity, 100%), and B. ambifaria (sensitivity and specificity, 100%) were the most efficient. However, the B. cepacia genomovar I assay lacked sensitivity (72%) and cross-reacted with all B. pyrrocinia isolates examined. Several new recA RFLP types were also revealed within the B. cepacia complex. One of these profiles was shared by a clinical and an environmental B. cepacia-like isolate and by the B. ubonensis type strain. The latter organism is a recently described soil bacterium. Its relationship to the various B. cepacia complex genomovars needs further study.

Asunto(s)

Infecciones por Burkholderia/microbiología , Burkholderia cepacia/genética , Fibrosis Quística/microbiología , Rec A Recombinasas/genética , Infecciones del Sistema Respiratorio/microbiología , Asia , Australia , Técnicas de Tipificación Bacteriana/métodos , Burkholderia cepacia/clasificación , Cartilla de ADN , Europa (Continente) , Humanos , Datos de Secuencia Molecular , América del Norte , Filogenia , Reacción en Cadena de la Polimerasa/métodos , América del Sur , Especificidad de la Especie

RESUMEN

Burkholderia cepacia colonizes cystic fibrosis (CF) patients. We evaluated the impact of the use of a selective medium in the rate of B. cepacia recovery from respiratory samples of CF patients. During a 6-month period, respiratory samples were collected from 106 CF patients and cultivated on selective media including a B. cepacia selective medium. Confirmation of the identity of B. cepacia isolates was carried out by species specific PCR and determination of genomovar status performed by a sequential PCR approach. Results of B. cepacia isolation during this period were compared to the preceding two years, when the sample processing was identical except for the lack of the B. cepacia selective medium. B. cepacia was isolated in 11/257 (4.2%) of the samples using the selective medium, in contrast with the preceding two years, when it was isolated in 6/1029 samples (0.58%), p < 0.0001. Identity of all 11 isolates was confirmed by PCR and genomovar determination was accomplished in all but one isolate. These results suggest that the use of a selective medium increases recovery rate of B. cepacia from respiratory samples.

Asunto(s)

Burkholderia cepacia/aislamiento & purificación , Medios de Cultivo , Fibrosis Quística/microbiología , Esputo/microbiología , Adolescente , Adulto , Burkholderia cepacia/genética , Distribución de Chi-Cuadrado , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Reacción en Cadena de la Polimerasa , Especificidad de la Especie , Factores de Tiempo

RESUMEN

OBJECTIVES: We sought to determine whether the same Burkholderia cepacia complex strain has persisted as the dominant clonal lineage among patients in a large cystic fibrosis (CF) treatment center during the past 2 decades. STUDY DESIGN: The inter-city spread of B cepacia through transfer of a colonized patient and the impact of infection control measures in containing inter-patient transmission were investigated. We analyzed all available B cepacia complex isolates recovered from 1981 to 1987 and from 1996 to 2000 at one large CF treatment center (Center A) and from 1997 to 2000 at another center (Center B). Incidence of B cepacia complex infection and infection control measures in both centers were assessed. RESULTS: Seventeen (81%) of 21 Center A patients from whom B cepacia complex bacteria were recovered between 1981 and 1987 and 40 (97%) of 41 patients culture-positive between 1996 and 2000 were infected with the same genomovar III strain. Transfer of a colonized patient from Center A to Center B was associated with an increase in B cepacia complex infection in Center B, all of which was with the Center A dominant strain. This strain, designated PHDC, lacks both B cepacia epidemic strain and cblA markers. CONCLUSIONS: B cepacia complex strains may remain endemic in CF treatment centers for many years. Responsible bacterial and host factors and optimal infection control measures to prevent inter-patient spread remain to be identified.

Asunto(s)

Infecciones por Burkholderia/transmisión , Burkholderia cepacia/clasificación , Burkholderia cepacia/genética , Fibrosis Quística/microbiología , Técnicas de Tipificación Bacteriana , Infecciones por Burkholderia/genética , Infecciones por Burkholderia/prevención & control , Genotipo , Humanos , Esputo/microbiología , Población Urbana

RESUMEN

In the genome of Burkholderia cepacia strain IPT64, which accumulates a blend of the two homopolyesters poly(3-hydroxybutyrate), poly(3HB), and poly(3-hydroxy-4-pentenoic acid), poly(3H4PE), from sucrose or gluconate as single carbon source, the polyhydroxyalkanoate (PHA) synthase structural gene was disrupted by the insertion of a chloramphenicol-resistant gene cassette (phaC1::Cm). The suicide vector pSUP202 harboring phaC1::Cm was transferred to B. cepacia by conjugation. The inactivated gene was integrated into the chromosome of B. cepacia by homologous recombination. This mutant and also 15 N-methyl-N'-nitrosoguanidine (NMG)-induced mutants still accumulated low amounts of PHAs and expressed low PHA synthase activity. The analysis of the mutant phaC1::Cm showed that it accumulated about 1% of PHA consisting of 68.2 mol% 3HB and 31.8 mol% 3H4PE from gluconate. The wild-type, in contrast, accumulated 49.3% of PHA consisting of 96.5 mol% 3HB and 3. 5 mol% 3H4PE. Our results indicated that the genome of B. cepacia possesses at least two PHA synthase genes, which probably have different substrate specificities.

Asunto(s)

Aciltransferasas/genética , Burkholderia cepacia/genética , Burkholderia cepacia/metabolismo , Genes Bacterianos , Poliésteres/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Aciltransferasas/metabolismo , Southern Blotting , Genoma Bacteriano , Hidroxibutiratos/metabolismo , Mutación , Ácidos Pentanoicos/metabolismo , Recombinación Genética , Especificidad por Sustrato , Valeratos/metabolismo

RESUMEN

A genomic library from Burkholderia cepacia IS-16 was constructed in Escherichia coli by partial Sau3AI digestion of the chromosomal DNA, with the plasmid vector Bluescript SK. This library was screened for clones able to grow as green stained colonies on selective medium developed for detecting phosphatase-positive colonies. Three green-stained clones (pFS1, pFS2, and pFS3) carried recombinant plasmids harboring DNA inserts of 5.0, 8.0, and 0.9 kb, respectively. DNA hybridization experiments demonstrated the presence of overlapping DNA fragments in the three clones and that these three clones were all derived from Burkholderia cepacia IS-16 genomic DNA. DNA sequence analysis, together with polyacrylamide gels of proteins encoded by E. coli containing pFS3, suggested that the isolated 0. 9-kb DNA fragment encodes the functional portion of a phosphate transport protein.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/genética , Burkholderia cepacia/genética , Genes Bacterianos , Monoéster Fosfórico Hidrolasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Burkholderia cepacia/enzimología , Escherichia coli/genética , Biblioteca de Genes , Vectores Genéticos , Datos de Secuencia Molecular , Plásmidos

RESUMEN

To assess the risk of acquisition of Pseudomonas cepacia by person-to-person transmission at cystic fibrosis summer camps, we conducted in 1990 a study at three camps attended by patients with cystic fibrosis who had P. cepacia infection and patients without P. cepacia infection but who were considered susceptible to infection. We obtained sputum or throat cultures from campers on their arrival at, weekly during, at the end of, and 14 to 30 days after camp. We compared the incidence of sputum conversion of patients at camp with that of patients outside camp by culturing specimens from noncamper control subjects with cystic fibrosis who were known not to be infected < or = 2 weeks before and 4 to 6 weeks after camp. We also determined the risk factors for P. cepacia acquisition by determining the relative risk of acquisition between campers who were exposed versus campers who were not exposed to campers known to be infected or to potential environmental sources of P. cepacia at camp. The ribotype of P. cepacia isolates from campers with sputum conversion was compared with that of isolates from other campers and from an environmental source. The cumulative incidence of sputum conversion during the study period was 6.1% (11/181) among campers compared with no incidence (0/92) among noncampers (p = 0.02, Fisher Exact Test). The incidence of sputum conversion at camp varied according to the prevalence of campers with known infection (p < 0.001, chi-square test for trend). The rate of sputum conversion was higher in the camp with longer duration (relative risk = 12.0; 95% confidence interval = 2.7 to 53.5). Ribotyping showed that P. cepacia isolates from all 11 campers with sputum conversion were identical or similar (1 to 2 band difference) to isolates of other P. cepacia-infected campers including co-converters. These results suggest that P. cepacia can be acquired by patients with cystic fibrosis who are attending summer camp for such patients, possibly through person-to-person transmission, and that the risk increases with the prevalence of P. cepacia-infected campers and the duration of camp.

Asunto(s)

Burkholderia cepacia , Acampada , Fibrosis Quística/complicaciones , Infecciones por Pseudomonas/transmisión , Adolescente , Adulto , Técnicas de Tipificación Bacteriana , Burkholderia cepacia/clasificación , Burkholderia cepacia/genética , Burkholderia cepacia/aislamiento & purificación , Estudios de Casos y Controles , Niño , ADN Bacteriano/análisis , Femenino , Humanos , Incidencia , Masculino , Polimorfismo de Longitud del Fragmento de Restricción , Infecciones por Pseudomonas/epidemiología , Infecciones por Pseudomonas/microbiología , Factores de Riesgo , Esputo/microbiología