Co-existence of blaOXA-23 and blaVIM in carbapenem-resistant Acinetobacter baumannii isolates belonging to global complex 2 in a Chinese teaching hospital / 中华医学杂志(英文版)

Background:@#Carbapenem-resistant Acinetobacter baumannii (CRAB) have been a challenging concern of health-care associated infections. The aim of the current study was to investigate the molecular epidemiology and clonal dissemination of CRAB isolates in a Chinese teaching hospital.@*Methods:@#Non-duplicate clinical A. baumannii isolates were collected from inpatients, and we measured the minimal inhibitory concentrations to determine antimicrobial susceptibility. Polymerase chain reaction (PCR) and sequencing were performed to detect carbapenem-resistance genes and occurrence of transposons among CRAB isolates. Moreover, the genetic diversity among isolates and clonal dissemination were determined by repetitive element PCR-mediated DNA fingerprinting (rep-PCR) and multilocus sequence typing (MLST).@*Results:@#A total of 67 CRAB isolates displayed resistance to most of the antibiotics tested in this study, except tigecycline. We detected blaOXA-23, blaOXA-51, blaOXA-58, and blaVIM genes in 94.0%, 100.0%, 1.5%, and 80.6% of the CRAB isolates, respectively. Nevertheless, 74.6% of the CRAB isolates co-harbored the blaOXA-23 and blaVIM. Only one type of transposons was detected: Tn2008 (79.1%, 53/67). Although 12 distinctive types (A-L) were determined (primarily A type) ST195 was the most prevalent sequence type (ST). ST368, ST210, ST90, ST829, and ST136 were also detected, and all belonged to clonal complex 208 (CC208) and global complex 2 (GC2).@*Conclusion:@#The blaOXA-23 and blaVIM genes contributed to the resistance among CRAB isolates collected in our study. Notably, most of the CRAB strains co-harbored blaOXA-23 and blaVIM genes, as well as Tn2008, which could contribute to clonal dissemination. The prevalence of such organisms may underlie hospital acquired infections.

Rapid Detection of Pseudomonas aeruginosa and Acinetobacter baumannii Harboring blaVIM-2, blaIMP-1 and blaOXA-23 Genes by Using Loop-Mediated Isothermal Amplification Methods

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla OXA-23, which are critical components for carbapenem resistance. METHODS: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO4) by testing different concentrations of MgSO4. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates. RESULTS: Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles. CONCLUSIONS: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.

Molecular characterization of metallo β-lactamase producing multidrug resistant Pseudomonas aeruginosa from various clinical samples.

Introduction: Pseudomonas aeruginosa is a potent opportunistic nosocomial human pathogen among Gram-negative bacteria causing various life-threatening infections in patients from Intensive Care Units. This bacterium has become resistant to almost all commonly available antibiotics with limited treatment options. Multi drug resistant P. aeruginosa (MDRPA) is a major cause of concern among hospital acquired infections. It uses distinctive resistant mechanisms virtually to all the available antibiotics such as Metallo β-lactamases (MBL) production, extended spectrum β-lactamase production (ESBL), up regulation of effl ux systems related genes and decreased outer membrane permeability. This study was carried out to fi nd one the predominant resistance mechanisms among MDRPA and the prevalence of corresponding resistance genes. Materials and Methods: MDRPA isolates collected from various clinical samples for a period of 1-year (November 2009-Octo ber 2010) were included to detect the predominant mechanism of resistance using phenotypic and molecular methods. Molecular characterization of all these isolates was done by polymerase chain reaction (PCR) for the presence of blaVIM-2, blaIMP-1, blaOXA-23, and blaNDM-1 genes with specifi c primers. Results: Among 75 MDRPA isolates 84% (63) were MBL producers. Molecular characterization studied by PCR showed the presence of blaVIM-2 gene in 13% of MBL producers. Conclusion: The prevalence of MBLs has been increasing worldwide, particularly among P. aeruginosa, leading to severe limitations in the therapeutic options for the management. Thus, proper resistance screening measures and appropriate antibiotic policy can be strictly adopted by all the healthcare facility providers to overcome these superbugs.

Emergence of VIM-2 metallo-beta-lactamase producing Ralstonia pickettii clinical isolate in India.

A multidrug‑resistant clinical isolate of Ralstonia pickettii from a woman was analysed. Modified Hodge test was positive for carbapenemase production. Conjugation experiment revealed the presence of conjugative plasmid of >140 Kb size typed as IncN type. This is the first report of emergence blaVIM‑2 in R. pickettii in India.

Evaluación de métodos fenotípicos para la detección de metalobetalactamasas en aislados clínicos de Pseudomonas aeruginosa / Assessment of Phenotypic Methods for Metalobetalactamase Detection in Clinical Isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa es considerado uno de los patógenos nosocomiales más importantes, siendo común su aislamiento en pacientes de unidades de cuidados intensivos, en quienes además suele presentar multirresistencia, esto limita enormemente las opciones terapéuticas para el tratamiento del paciente, lo que constituye un problema ya que no se disponen de drogas efectivas para el tratamiento. El presente trabajo tiene como objetivo evaluar cuatro métodos fenotípicos para la detección de metalo-beta-lactamasas (método de sinergia del doble disco con EDTA, método del disco combinado de imipenem y meropenem con EDTA, método de E-Test-MBL y el método de Hodge modificado), con un método de referencia (PCR), para determinar el método fenotípico o combinación de estos que sea más valido y seguro para su implementación en los laboratorios de bacteriología de rutina. La sensibilidad y especificidad de los métodos fenotípicos empleados fue buena (más de 90%), de los métodos empleados, el test de Hodge modificado fue el que proporcionó mejores resultados en la detección de aislados de P. aeruginosa productores de MBL (menor tasa de falsos positivos y negativos), al analizar los diferentes métodos combinados para determinar cuál es la asociación con mayor sensibilidad y especificidad, el método del doble disco asociado con el test de Hodge modificado reunieron estas características, estos resultados sugieren que estos dos métodos se deben emplear de rutina en un laboratorio de Bacteriología, por ser fáciles de realizar, económicos, sensibles y específicos.

Pseudomonas aeruginosa is considered one of the most important nosocomial pathogens. Its isolation is commonin intensive care unit patients who also usually have multi-resistance. This greatly limits therapeutic options, since no effective drugs are available for treatment. The study aims to evaluate four phenotypic methods for detecting metallo-beta-lactamases. The double disc synergy with EDTA method, combined imipenem and meropenem disk with EDTA method, the E-Test-MBL method and the modified Hodge method, along with a reference method (PCR), are used to determine the phenotypic method or a combination of these is used that is more valid and safe for implementation in routine bacteriology laboratories. The sensitivity and specificity of the phenotypic methods used was good (over 90%); the modified Hodge test obtained the best results in detecting MBL-producing P. aeruginosa isolates (lower rate of false positive and negative). On analyzing the different combined methods for determining the association with greater sensitivity and specificity, the double disc method associated with the modified Hodge test met these characteristics. Results suggest that these two methods should be routinely used in a bacteriology laboratory, since they are easy to perform, sensitive, specific and economical.

Detection and characterization of metallo-β-lactamases in Pseudomonas aeruginosa by phenotypic and molecular methods from clinical samples in a tertiary care hospital / Detección y caracterización de la metallo-betalactamasa en la Pseudomonas aeruginosa mediante métodos fenotípicos y moleculares a partir de muestras clínicas en un hospital de atención terciaria

AIMS: The aim of this study was to detect and characterize the presence of metallo-β-lactamase (MBL) production in multidrug resistant (MDR) P aeruginosa collected from clinical samples in a tertiary care hospital. METHODS AND MATERIALS: A total of 67 non-repetitive isolates of MDR P aeruginosa recovered from various clinical specimens were screened for MBL production by IPM/MEM-EDTA combined disc test. Polymerase chain reaction was performed on all isolates using blaIMP and blaVIM consensus primers to characterize them genotypically. RESULTS: Among 67 P aeruginosa isolates, 62.7% (42/67) and 70.1% (47/67) were resistant to imipenem and meropenem respectively and 47 (70.1%) were found to be MBL producers. Among this 47 MBL-producing isolates, 41 (61.1%) strains carried the blaVIM gene and 2 (3%) strains carried the blaIMP gene. Three strains were phenotypically negative but positive genotypically for blaVIM gene. One strain was resistant to both imipenem and meropenem but did not show phenotypic positivity. CONCLUSION: This study confirms the dissemination of blaVIM genes among MDR Pseudomonas aeruginosa and hence it is indispensible to identify and aptly control the threat of horizontal and vertical transfer.

OBJETIVO: El objetivo de este estudio es descubrir y caracterizar la presencia de producción de metallo-betalactamasa (MBL) en P aeruginosa resistente a los multifármacos (RMF), recogida de muestras clínicas de un hospital de atención terciaria. MÉTODO: Un total de 67 aislados no repetitivos de P aeruginosa RMF obtenidos de varios specímenes clínicos, fueron tamizados en busca de producción de MBL, mediante una prueba de disco combinado IPM/MEM-EDTA. Se efectuó una reacción en cadena de la polimerasa sobre todos los aislados, usando iniciadores de consenso blaIMP y blaVIM para la caracterización genotípica. RESULTADOS: Entre los aislados de P aeruginosa, 62.7% (42/67) y 70.1% (47/67) fueron resistentes al Imipenem y al Meropenem respectivamente, mientras que se halló que 47 (70.1%) eran productores de MBL. De los 47 aislados productores de MBL, 41 (61.1%) cepas eran portadoras del gen blaVIM en tanto que 2 (3%) cepas eran portadoras del gen blaIMP. Tres cepas fueron fenotípicamente negativas, pero genotípicamente positivas con respecto al gen blaVIM. Una cepa fue resistente tanto al Imipenem como al Meropenem, pero no mostró positividad fenotípicamente. CONCLUSIÓN: El presente estudio confirma la diseminación de los genes blaVIM entre las Pseudomonas aeruginosa RMF. Es importante identificar así como controlar adecuadamente la amenaza de la transferencia horizontal y vertical.

Detección del gen blaVIM-2 en cepas de Pseudomonas aeruginosa productoras de metalo ß-lactamasa aisladas en una unidad de cuidados intensivos en Ciudad Bolívar, Venezuela / blaVIM-2 gene detection in metallo-ß-lactamase-producing Pseudomonas aeruginosa strains isolated in an Intensive Care Unit in Ciudad Bolívar, Venezuela

Ten Pseudomonas aeruginosa strains with resistance to broad-spectrum cephalosporin and carbapenems were studied to determine the presence of genes that mediate the productionof metallo-p-lactamases. These strains were isolated from patients with nosocomial infection at the Intensive Care Unit of the Complejo Hospitalario "Ruiz y Paéz" of Ciudad Bolívar, Bolívar State, Venezuela, from 2003 to 2006. In all isolates a metallo-enzyme activity was detected by using the double disk synergism test. PCR amplification of genes encoding the families IMP, VIM and SPM metallo-ß-lactamases showed the presence of a blaVIM gene in all strains studied. DNA sequencing revealed that all isolates showed the presence of blaVIM-2These results suggest that it is necessary to keep these strains under epidemiologic surveillance, establish laboratory strategies for opportune detection and the imple-mentation of new policies to ensure the appropriate use of antibiotics in this institution.

Se estudiaron 10 cepas de Pseudomonas aeruginosa con resistencia a cefalosporinas y carbapenémicos con el objeto de determinar la presencia de genes que median la producción de metalo ß-lactamasas. Estas cepas se aislaron de pacientes con infección nosocomial hospitalizados en la Unidad de Cuidados Intensivos (UCI) del Complejo Hospitalario "Ruiz y Paéz" de Ciudad Bolívar, estado Bolívar, Venezuela, durante el período 2003 - 2006. En todas las cepas se detectó la actividad de una metalo-enzima, mediante la prueba del sinergismo del doble disco. La amplificación por la reacción de polimerasa en cadena de los genes que codifican para las metalo ß-lactamasas de las familias IMP, VIM y SPM, y su posterior secuenciación, permitió confirmar la presencia de metalo p-lactamasas de tipo VIM-2. Estos resultados sugieren que es necesario mantener bajo vigilancia epidemiológica a estas cepas, establecer estrategias de laboratorio para su detección oportuna e implementar nuevas políticas que aseguren el uso adecuado de los antibacterianos en esta institución.

Antimicrobial Resistance of Clinical Isolates of Acinetobacter spp. Collected from Non-Tertiary Hospitals and Detection of a Metallo-beta-Lactamase-Producing Strain / 감염과화학요법

BACKGROUND: The aim of this study was to investigate the antimicrobial resistance of clinical isolates of Acinetobacter spp. collected from non-tertiary hospitals and to characterize the phenotype and the genotype of imipenem-non-susceptible isolates. MATERIALS AND METHODS: Clinical isolates of Acinetobacter spp. were identified using recA-restriction fragment length polymorphism (RFLP) analysis with Tsp5091. Susceptibility to antimicrobial agents was determined using disk diffusion test and agar dilution test according to the criteria of the National Committee for Clinical Laboratory Standards. PCR and sequence analyses were used to detect the blaIMP-1 and blaVIM-2 genes, and to determine the content and order of the resistance genes inserted in integron. RESULTS: Of 71 Acinetobacter spp. isolates collected from non-tertiary hospitals during 2002 and 2003, 60 isolates were A. baumannii, and 2, 4, and 5 isolates were Acinetobacter genomic species 3, 13TU, and A. lwoffii, respectively. The resistance rate of Acinetobacter spp. isolates to beta-lactams, aminoglycosides, and fluoroquinolones was high except for imipenem and meropenem. The presence of blaVIM-2 gene was found in one isolate, Acinetobacter genomic species 13TU, for which the MIC of imipenem was 8 mg/L; the blaVIM-2 gene of this strain was located on 3 kb class 1 integron with aacA7 and aadA1 genes. CONCLUSIONS: Among the tested agents, imipenem and meropenem retained greatest activity against Acinetobacter spp. isolates collected from non-tertiary hospitals. This is the first report of VIM-2-producing Acinetobacter genomic species 13TU strains with class 1 integron containing blaVIM-2 gene.

Antimicrobial Resistance of Clinical Isolates of Acinetobacter spp. Collected from Non-Tertiary Hospitals and Detection of a Metallo-beta-Lactamase-Producing Strain / 감염과화학요법

BACKGROUND: The aim of this study was to investigate the antimicrobial resistance of clinical isolates of Acinetobacter spp. collected from non-tertiary hospitals and to characterize the phenotype and the genotype of imipenem-non-susceptible isolates. MATERIALS AND METHODS: Clinical isolates of Acinetobacter spp. were identified using recA-restriction fragment length polymorphism (RFLP) analysis with Tsp5091. Susceptibility to antimicrobial agents was determined using disk diffusion test and agar dilution test according to the criteria of the National Committee for Clinical Laboratory Standards. PCR and sequence analyses were used to detect the blaIMP-1 and blaVIM-2 genes, and to determine the content and order of the resistance genes inserted in integron. RESULTS: Of 71 Acinetobacter spp. isolates collected from non-tertiary hospitals during 2002 and 2003, 60 isolates were A. baumannii, and 2, 4, and 5 isolates were Acinetobacter genomic species 3, 13TU, and A. lwoffii, respectively. The resistance rate of Acinetobacter spp. isolates to beta-lactams, aminoglycosides, and fluoroquinolones was high except for imipenem and meropenem. The presence of blaVIM-2 gene was found in one isolate, Acinetobacter genomic species 13TU, for which the MIC of imipenem was 8 mg/L; the blaVIM-2 gene of this strain was located on 3 kb class 1 integron with aacA7 and aadA1 genes. CONCLUSIONS: Among the tested agents, imipenem and meropenem retained greatest activity against Acinetobacter spp. isolates collected from non-tertiary hospitals. This is the first report of VIM-2-producing Acinetobacter genomic species 13TU strains with class 1 integron containing blaVIM-2 gene.