RESUMEN
BACKGROUND: We investigated an outbreak of multidrug-resistant Acinetobacter baumannii-calcoaceticus complex infection among US service members injured in Iraq. METHODS: The investigation was conducted in Iraq and Kuwait, in the 2 military hospitals where the majority of injured service members were initially treated. After initially characterizing the outbreak, we evaluated 3 potential sources of infection for the period March 2003 to December 2004. The evaluation included screening samples that were obtained from the skin of patients for the presence of colonization and assessing the soil and health care environments for the presence of A. baumanii-calcoaceticus complex organisms. Isolates obtained from samples from patients in US Military treatment facilities, as well as environmental isolates, were genotypically characterized and compared using pulsed-field gel electrophoresis. RESULTS: A. baumanii-calcoaceticus complex organisms were present on the skin in only 1 (0.6%) of 160 patients who were screened and in 1 (2%) of 49 soil samples. A. baumanii-calcoaceticus complex isolates were recovered from treatment areas in 7 of the 7 field hospitals sampled. Using pulsed-field gel electrophoresis, we identified 5 cluster groups in which isolates from patients were related to environmental isolates. One cluster included hospitalized patients who had not been deployed to Iraq. Among the clinical isolates, only imipenem, polymyxin B, and colistin demonstrated reliable in vitro antimicrobial activity. Generally, the environmental isolates were more drug susceptible than were the clinical isolates. CONCLUSIONS: Our findings suggest that environmental contamination of field hospitals and infection transmission within health care facilities played a major role in this outbreak. On the basis of these findings, maintaining infection control throughout the military health care system is essential. Novel strategies may be required to prevent the transmission of pathogens in combat field hospitals.
Asunto(s)
Infecciones por Acinetobacter/epidemiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter calcoaceticus/efectos de los fármacos , Infección Hospitalaria/microbiología , Brotes de Enfermedades , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Contaminación de Equipos , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/transmisión , Acinetobacter baumannii/genética , Acinetobacter baumannii/patogenicidad , Acinetobacter calcoaceticus/genética , Acinetobacter calcoaceticus/patogenicidad , Adulto , Infección Hospitalaria/epidemiología , Infección Hospitalaria/transmisión , Electroforesis en Gel de Campo Pulsado , Exposición a Riesgos Ambientales , Femenino , Hospitales Militares , Humanos , Control de Infecciones/métodos , Irak/epidemiología , Kuwait/epidemiología , Masculino , Pruebas de Sensibilidad Microbiana , Personal Militar , Epidemiología Molecular , Filogenia , Estados UnidosRESUMEN
In an effort to find a rapid, efficient, and reliable method for screening and classifying large numbers of tetracycline-resistant bacterial isolates, we developed a multiplex, real-time PCR assay using SYBR Green I and the Roche LightCycler. The assay can rapidly identify eight genes encoding tetracycline resistance efflux pumps including tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H) and tet(J). Primers were selected for PCR amplification of these eight tetracycline resistance determinant (tet) genes commonly found in Gram-negative organisms. We combined primer pairs together to make a single-tube multiplex PCR reaction followed by melting curve analysis. Amplification of the expected tet gene products was confirmed by both agarose gel electrophoresis and DNA sequence analysis. Based on melting temperature differences, we could identify the different classes of tet genes. To test the multiplex PCR, the assay was used on 107 tetracycline-resistant clinical isolates of various Gram-negative organisms isolated in several locations around the world. About 49.5% of those strains carried a tet(A) gene, 35.5% carried a tet(B), 7.5% carried a tet(J), 5.6% carried a tet(C) and 1.9% carried a tet(D) gene. DNA sequence analysis of the amplicons confirmed that the specificity of the test was 100%. The sensitivity of the multiplex test varied from 10 to 1000 CFU per PCR reaction. Our real time PCR assay utilizing SYBR Green I and melting point analysis on the Lightcycler system showed not only a high confidence level in differentiation of the classes of tet genes but also precise reproducibility. Our multiplex PCR tet gene class identification assay offers a significant savings of time and labor in the analysis of large numbers of clinical strains compared with assays using individual gene PCR or traditional phenotype methods.
Asunto(s)
ADN Bacteriano/análisis , ADN Bacteriano/genética , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/aislamiento & purificación , Compuestos Orgánicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Resistencia a la Tetraciclina/genética , Benzotiazoles , Cartilla de ADN/metabolismo , Diaminas , Fluorescencia , Genes Bacterianos , Desnaturalización de Ácido Nucleico , Quinolinas , Reproducibilidad de los Resultados , Temperatura de TransiciónRESUMEN
Members of the genus Acinetobacter are ubiquitous in soil and water and are an important cause of nosocomial infections. A rapid method is needed to genotype Acinetobacter isolates to determine epidemiology and clonality during infectious outbreaks. Multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) is a method that uses the amplicon base compositions to genotype bacterial species. In order to identify regions of the Acinetobacter genome useful for this method, we sequenced regions of six housekeeping genes (trpE, adk, efp, mutY, fumC, and ppa) from 267 isolates of Acinetobacter. Isolates were collected from infected and colonized soldiers and civilians involved in an outbreak in the military health care system associated with the conflict in Iraq, from previously characterized outbreaks in European hospitals, and from culture collections. Most of the isolates from the Iraqi conflict were Acinetobacter baumannii (189 of 216 isolates). Among these, 111 isolates had genotypes identical or very similar to those associated with well-characterized A. baumannii isolates from European hospitals. Twenty-seven isolates from the conflict were found to have genotypes representing different Acinetobacter species, including 8 representatives of Acinetobacter genomospecies 13TU and 13 representatives of Acinetobacter genomospecies 3. Analysis by the PCR/ESI-MS method using nine primer pairs targeting the most information-rich regions of the trpE, adk, mutY, fumC, and ppa genes distinguished 47 of the 48 A. baumannii genotypes identified by sequencing and identified at the species level at least 18 Acinetobacter species. Results obtained with our genotyping method were essentially in agreement with those obtained by pulse-field gel electrophoresis analysis. The PCR/ESI-MS genotyping method required 4 h of analysis time to first answer with additional samples subsequently analyzed every 10 min. This rapid analysis allows tracking of transmission for the implementation of appropriate infection control measures on a time scale previously not achievable.