RESUMEN

Pseudomonas infections are an important cause of morbidity and mortality in immunocompromised patients. We present here data for the spread of Pseudomonas fluorescens caused by a contaminated drinking water dispenser in a bone marrow transplant unit. Over a 1-month period we observed a sharp increase in the isolation of P. fluorescens from weekly pharyngeal surveillance swabs. Environmental samples were taken from a variety of water sources throughout the unit. These samples were cultured on cetrimide agar medium, and isolates were epidemiologically characterized by antibiotic susceptibility patterns and molecular typing methods. Nine patients became colonized with P. fluorescens, and six out of the nine developed febrile neutropenia. P. fluorescens was cultured after the filtration of 100 ml of drinking water from one of two stand-alone chiller units supplying cooled bottled water to the bone marrow transplant unit. All other environmental samples were negative. There were no further cases of P. fluorescens colonization after the contaminated dispenser was removed. Molecular typing showed that all P. fluorescens isolates were identical by both random amplification of polymorphic DNA PCR and pulsed-field gel electrophoresis. We recommend that such bottled water supplies not be used in high-risk areas or be subject to regular microbiological monitoring.

Asunto(s)

Infección Hospitalaria/epidemiología , Infecciones por Pseudomonas/epidemiología , Pseudomonas fluorescens/aislamiento & purificación , Microbiología del Agua , Adulto , Técnicas de Tipificación Bacteriana , Técnicas Bacteriológicas , Portador Sano/epidemiología , Portador Sano/microbiología , Portador Sano/transmisión , Análisis por Conglomerados , Infección Hospitalaria/microbiología , Infección Hospitalaria/transmisión , Electroforesis en Gel de Campo Pulsado , Femenino , Fiebre de Origen Desconocido/epidemiología , Fiebre de Origen Desconocido/microbiología , Genotipo , Hospitales , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Epidemiología Molecular , Tipificación Molecular , Faringe/microbiología , Infecciones por Pseudomonas/transmisión , Técnica del ADN Polimorfo Amplificado Aleatorio , Adulto Joven

RESUMEN

Ninety-six genetically diverse multidrug-resistant clinical isolates of Acinetobacter baumannii from 25 hospitals in 17 European countries were screened by PCR for specific carbapenemase-hydrolyzing class D ß-lactamase (CHDL) genes and by PCR-based replicon typing for the presence of 19 different plasmid replicase (rep) gene homology groups (GRs). Results were confirmed by DNA sequencing where necessary. All 96 isolates contained at least 1 (with a maximum of 4) of the 19 groups of rep genes. Groups detected were GR6 (repAci6; 93 isolates), GR2 (including repAci1 [67 isolates] and repAci2 [3 isolates]), GR16 (repApAB49; 12 isolates), GR12 (p2ABSDF0001; 10 isolates), GR3 (repAci3; 4 isolates), GR4 (repAci4; 3 isolates), GR10 (repAciX; 1 isolate), and GR14 (repp4AYE; 1 isolate). Variations in rep gene content were observed even among epidemiologically related isolates. Genes encoding OXA-58-like CHDLs (22 isolates) were associated with carriage of the repAci1, repAci3, repAci4, and repAciX genes, genes encoding OXA-40-like CHDLs (6 isolates) were associated with repAci2 and p2ABSDF0001, and genes encoding OXA-23-like CHDLs (8 isolates) were associated with repAci1. Most intrinsic Acinetobacter plasmids are non-self-transferable, but the almost ubiquitous repAci6 gene was strongly associated with a potential tra locus that could serve as a general system for plasmid mobilization and consequent horizontal transmission of plasmids and their associated antibiotic resistance genes among strains of A. baumannii.

Asunto(s)

Acinetobacter baumannii/enzimología , Acinetobacter baumannii/genética , Carbapenémicos/metabolismo , Plásmidos/genética , beta-Lactamasas/metabolismo , beta-Lactamasas/genética

RESUMEN

OBJECTIVES: The aim of the study was to determine whether ATPase genes of genetically diverse Acinetobacter baumannii isolates are disrupted by potential genomic islands. METHODS: Random amplified polymorphic DNA (RAPD)-PCR, sequence grouping and PFGE were used to investigate the genetic diversity of 50 A. baumannii isolated from various clinical specimens. PCR analysis was then used to identify isolates with a potentially disrupted ATPase gene. Representative genetically distinct isolates were further characterized by PCR mapping and chromosome walking to analyse the flanking regions of the disrupted ATPase genes. RESULTS: Forty-one of the 50 isolates tested appeared to contain a disrupted ATPase gene. Sequence group and PFGE data for 10 ATPase PCR-negative representative isolates confirmed substantial genetic diversity. Seven isolates contained elements with ends showing high levels of sequence similarity to one or both extremities of AbaR1, the first resistance island described in A. baumannii. A further isolate, A25, possessed a highly conserved AbaR1-like 3'-end, but a divergent, though related, 5'-terminus that exhibited near identity with a distinct locus in A. baumannii ATCC 17978. A ninth isolate (A92) possessed a completely novel sequence abutting on its 5'-ATPase remnant. Three isolates appeared to lack 3'-ATPase gene segments, as was the case with the recently sequenced strain ACICU. Thus, 8 of the 10 ATPase-negative isolates investigated in detail had ATPase genes disrupted with AbaR1-like flanking regions. CONCLUSIONS: ATPase genes of diverse A. baumannii isolates are frequently disrupted by insertions matching AbaR1-related flanking sequences. However, the ATPase gene of isolate A92 was disrupted by a DNA sequence distinct from those found in AbaR1.

Asunto(s)

Acinetobacter baumannii/enzimología , Acinetobacter baumannii/genética , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , ADN Recombinante , Mutagénesis Insercional , Análisis por Conglomerados , Dermatoglifia del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Electroforesis en Gel de Campo Pulsado , Humanos , Datos de Secuencia Molecular , Polimorfismo Genético , Técnica del ADN Polimorfo Amplificado Aleatorio , Análisis de Secuencia de ADN

RESUMEN

A total of 200 blood cultures containing putative staphylococci were analyzed by a commercial gene probe hybridization assay (EVIGENE; Statens Serum Institut, Copenhagen, Denmark), and 18 were identified as methicillin-resistant Staphylococcus aureus (MRSA) positive. Of these, 17 were positive by PCR and 16 were positive by culture. Detailed analysis of the discrepant results showed that the EVIGENE kit allowed specific identification of MRSA in blood cultures without any of the drawbacks associated with PCR.

Asunto(s)

ADN Bacteriano/sangre , Resistencia a la Meticilina/fisiología , Staphylococcus aureus/aislamiento & purificación , Sondas de ADN , ADN Bacteriano/aislamiento & purificación , Humanos , Reacción en Cadena de la Polimerasa/métodos , Juego de Reactivos para Diagnóstico , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética

RESUMEN

A new molecular assay (CytAMP) utilizing isothermal signal-mediated amplification of RNA was evaluated for rapid detection of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA). The assay targeted the coa (coagulase) and mecA genes, thereby simultaneously identifying S. aureus and methicillin (oxacillin) resistance. Results were obtained in approximately 3.5 h as a color signal in 96-well microtiter plates. The detection limit was between 2 x 10(5) and 10(6) CFU/assay, equivalent to 4 x 10(6) to 2 x 10(7) CFU/ml in an overnight broth. This level of growth was obtained with an initial inoculum of 10 to 50 CFU. The CytAMP assay and a mecA-femB PCR assay both detected 113 MRSA strains among 396 clinical isolates of bacteria (CytAMP sensitivity and specificity were both 100% relative to those of PCR). Conventional culture detected 109 MRSA strains, but with 19 false-positive and 23 false-negative results relative to both molecular methods. Discrepancies were also observed for 100 enrichment broths containing MRSA screening swabs, with 11 broths culture negative but PCR positive. CytAMP and PCR were more in agreement, but six broths were CytAMP negative and PCR positive. Five of these contained 10(2) to 10(5) CFU/assay (below the CytAMP detection limit of 2 x 10(5) CFU/assay), and the sixth contained 10(6) CFU/assay. Overall, culture and CytAMP had similar sensitivities and specificities relative to those of PCR, but the CytAMP assay enabled swabs to be analyzed as a batch following overnight incubation in enrichment broth, with results reported before 12 noon the next day.

Asunto(s)

Resistencia a la Meticilina , Técnicas de Amplificación de Ácido Nucleico/métodos , Manejo de Especímenes/métodos , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/aislamiento & purificación , Proteínas Bacterianas/genética , Coagulasa/genética , Humanos , Tamizaje Masivo , Sondas de Oligonucleótidos , Oxacilina/farmacología , ARN Bacteriano/genética , Sensibilidad y Especificidad , Staphylococcus aureus/clasificación , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Factores de Tiempo