ABSTRACT
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.
Subject(s)
Acinetobacter baumannii/genetics , Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , Drug Resistance, Bacterial/genetics , Genes, Bacterial , Nucleic Acid Amplification Techniques , Pseudomonas aeruginosa/genetics , Sensitivity and SpecificityABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Adolescent , Adult , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Young Adult , Pseudomonas aeruginosa/enzymology , beta-Lactamases/genetics , Anti-Bacterial Agents/pharmacology , Cross Infection/microbiology , Disk Diffusion Antimicrobial Tests , Drug Resistance, Bacterial/genetics , Intensive Care Units , Phenotype , Polymerase Chain Reaction , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Venezuela , Young Adult , beta-Lactamases/biosynthesisABSTRACT
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.
Subject(s)
Acinetobacter , Agar , Aminoglycosides , Anti-Infective Agents , beta-Lactams , Diffusion , Fluoroquinolones , Genotype , Imipenem , Integrons , Phenotype , Polymerase Chain Reaction , Sequence AnalysisABSTRACT
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.