RESUMEN
Antibiotic susceptibility testing with the BD Phoenix system on bacterial cell pellets generated from blood culture broths using the Bruker MALDI Sepsityper kit was evaluated. Seventy-six Gram-negative isolates, including 12 with defined multi-resistant phenotypes, had antibiotic susceptibility testing (AST) performed by Phoenix on the cell pellet in parallel with conventional methods. In total, 1414/1444 (97.9â%) of susceptibility tests were concordant, with only 1 (0.07â%) very major error. This novel method has the potential to reduce the turnaround time for AST results by up to a day for Gram-negative bacteraemias.
Asunto(s)
Antibacterianos/farmacología , Bacteriemia/microbiología , Bacterias/efectos de los fármacos , Sangre/microbiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Automatización de Laboratorios/métodos , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Factores de TiempoRESUMEN
OBJECTIVE: Blood pressure (BP) cuffs are potential vectors for transmission of multi-resistant organisms (MROs). The present study aims to determine MRO colonisation rates in BP cuffs from areas of high patient flow as an assessment of the quality of disinfection and infection control practices. METHODS: BP cuffs in the ED, high dependency unit (HDU) and operating theatres (OT) were prospectively examined after routine disinfection procedures. Swabs collected from the inner and outer surfaces of BP cuffs during inter-patient intervals were plated onto replicate organism detection and counting, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) chromogenic agar plates to detect rates of bacterial, MRSA and VRE colonisation, respectively. RESULTS: High bacterial colonisation rates were detected in BP cuffs from all three areas. BP cuffs from OT were significantly less colonised compared with cuffs from HDU and ED; 76% versus 96% and 100% (P < 0.0001) for inner surfaces and 86% versus 98% and 100% (P < 0.0001) for outer surfaces, respectively. Equivalent or higher bacterial growth was observed on the inner surface compared with outer surface in 54%, 84% and 86% of BP cuffs from OT, HDU and ED, respectively. MRSA was detected in 3 of 150 (2%) swabs collected, but no VRE was detected. CONCLUSION: Although MRSA and VRE were infrequently isolated, current disinfection and infection control protocols need to be improved given the greater recovery of organisms from the inner compared with outer surfaces of BP cuffs.
Asunto(s)
Desinfección/normas , Resistencia a Múltiples Medicamentos , Enterococcus/aislamiento & purificación , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Esfigmomanometros/microbiología , Infección Hospitalaria/microbiología , Infección Hospitalaria/prevención & control , Enterococcus/efectos de los fármacos , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Estudios Prospectivos , Resistencia a la VancomicinaRESUMEN
Early appropriate antibiotic treatment reduces mortality in severe sepsis, but current methods to identify antibiotic resistance still generally rely on bacterial culture. Modern diagnostics promise rapid gene detection, but the apparent diversity of relevant resistance genes in Enterobacteriaceae is a problem. Local surveys and analysis of publicly available data sets suggested that the resistance gene pool is dominated by a relatively small subset of genes, with a very high positive predictive value for phenotype. In this study, 152 Escherichia coli and 115 Klebsiella pneumoniae consecutive isolates with a cefotaxime, ceftriaxone and/or ceftazidime minimum inhibitory concentration (MIC) of ≥ 2 µg/mL were collected from seven major hospitals in Sydney (Australia) in 2008-2009. Nearly all of those with a MIC in excess of European Committee on Antimicrobial Susceptibility Testing (EUCAST) resistance breakpoints contained one or more representatives of only seven gene types capable of explaining this phenotype, and this included 96% of those with a MIC ≥ 2 µg/mL to any one of these drugs. Similarly, 97% of associated gentamicin-non-susceptibility (MIC ≥ 8 µg/mL) could be explained by three gene types. In a country like Australia, with a background prevalence of resistance to third-generation cephalosporins of 5-10%, this equates to a negative predictive value of >99.5% for non-susceptibility and is therefore suitable for diagnostic application. This is an important proof-of-principle that should be tested in other geographic locations.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Cefalosporinas/farmacología , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Pool de Genes , Klebsiella pneumoniae/efectos de los fármacos , Australia , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Pruebas de Sensibilidad MicrobianaRESUMEN
The early initiation of targeted antibiotic therapy in patients with bacteraemia and septic shock impacts favourably on outcomes. Rapid methods are therefore increasingly employed for bacterial identification directly from positive blood culture bottles, but with variable success. We evaluated the performance of the Gram Positive 12 multiplex tandem PCR (MT-PCR) assay (AusDiagnostics; catalogue no. 6202, version 07) containing targets for the identification of staphylococci including Staphylococcus aureus, streptococci including Streptococcus pneumoniae, enterococci including Enterococcus faecalis and Enterococcus faecium and their common antibiotic resistance genes (mecA, vanA, vanB). A total of 673 aerobic and anaerobic blood culture broths demonstrating Gram-positive cocci on microscopy were analysed in parallel with traditional phenotypic methods. Amplification of the internal control was inhibited in 79/673 (11.7â%) samples; however, MT-PCR identification was in concordance with phenotypic identification to the genus level in 96.6â% (537/556) of the remaining monomicrobial specimens and to the species level, where applicable, in 100â% (172/172) of samples. MT-PCR identification for 94.7â% (36/38) of polymicrobial samples matched traditional phenotypic identification. Meticillin and vancomycin susceptibility results determined by MT-PCR in blood culture broths demonstrated complete agreement with those determined by phenotypic methods in all 143 Staphylococcus aureus isolates and eight E. faecium isolates, respectively. Gram-positive pathogens and their key antibiotic resistance markers were reliably identified with the MT-PCR assay within 3 h of a positive blood culture result.
Asunto(s)
Antibacterianos/farmacología , Bacteriemia/microbiología , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Bacterias Grampositivas/microbiología , Reacción en Cadena de la Polimerasa Multiplex , Bacteriemia/diagnóstico , Proteínas Bacterianas/genética , Ligasas de Carbono-Oxígeno/genética , ADN Bacteriano/genética , Enterococcus/clasificación , Enterococcus/efectos de los fármacos , Enterococcus/genética , Humanos , Meticilina/farmacología , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las Penicilinas , ARN Ribosómico 16S/genética , Choque Séptico/diagnóstico , Choque Séptico/microbiología , Staphylococcus/clasificación , Staphylococcus/efectos de los fármacos , Staphylococcus/genética , Streptococcus/clasificación , Streptococcus/efectos de los fármacos , Streptococcus/genética , Vancomicina/farmacología , Resistencia a la Vancomicina/genéticaAsunto(s)
Técnicas Bacteriológicas/métodos , Clostridioides difficile/clasificación , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/diagnóstico , Errores Diagnósticos , Técnicas de Diagnóstico Molecular/métodos , Proteínas Bacterianas/genética , Clostridioides difficile/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Heces/microbiología , Humanos , Datos de Secuencia Molecular , Proteínas Represoras/genética , Análisis de Secuencia de ADNRESUMEN
Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of bacteria from blood culture broths. We evaluate for the first time, the performance of the MALDI Sepsityper™ Kit and MS for the identification of bacteria compared to standard phenotypic methods using the manufacturer's specified bacterial identification criteria (spectral scores ≥1.700-1.999 and ≥2.000 indicated identification to genus and species level, respectively). Five hundred and seven positive blood culture broths were prospectively examined, of which 379 (74.8%; 358 monomicrobial, 21 polymicrobial) were identified by MALDI-TOF MS; 195 (100%) and 132 (67.7%) of 195 gram-positive; and 163 (100%) and 149 (91.4%) of 163 gram-negative organisms from monomicrobial blood cultures were correctly identified to genus and species level, respectively. Spectral scores <1.700 (no identification) were obtained in 128/507 (25.2%) positive blood culture broths, including 31.6% and 32.3% of gram-positive and polymicrobial blood cultures, respectively. Significantly more gram-negative organisms were identified compared to gram-positive organisms at species level (p<0.0001). Five blood cultures were misidentified, but at species level only; including four monomicrobial blood cultures with Streptococcus oralis/mitis that were misidentified as Streptococcus pneumoniae. Positive predictive values for the direct identification of both gram-positive and gram-negative bacteria from monomicrobial blood culture broths to genus level were 100%. A diagnostic algorithm for positive blood culture broths that incorporates gram staining and MALDI-TOF MS should identify the majority of pathogens, particularly to genus level.
Asunto(s)
Bacterias/aislamiento & purificación , Técnicas Bacteriológicas/métodos , Sepsis/microbiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Bacterias/clasificación , Bacterias Gramnegativas/clasificación , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Grampositivas/clasificación , Bacterias Grampositivas/aislamiento & purificación , Humanos , Estudios Prospectivos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Sepsis/sangre , Sepsis/diagnóstico , Especificidad de la EspecieRESUMEN
A novel gene cassette, aacA43, was identified in the aadB-aacA43-oxa10-smr2 cassette array in a class 1 integron. Like related aminoglycoside-(6')-acetyltransferases, AacA43 confers clinically relevant resistance to kanamycin, tobramycin, and some less-used aminoglycosides but not to gentamicin. Although transferable on an IncL/M plasmid, aacA43 was identified in only two different Klebsiella pneumoniae strains (14 isolates), one Escherichia coli strain (2 isolates), and one Enterobacter cloacae strain in a survey of patients in a Sydney intensive care unit in 2004-2005.
Asunto(s)
Acetiltransferasas/genética , Integrones , Plásmidos , Secuencia de Aminoácidos , Secuencia de Bases , Farmacorresistencia Bacteriana , Enterobacter cloacae/genética , Escherichia coli/genética , Humanos , Klebsiella pneumoniae/genética , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
AIM: To evaluate the effect of defined mutations in the major OmpK35 and OmpK36 porins in Klebsiella pneumoniae on the activity of two common plasmid-mediated AmpC enzymes. METHODS: Naturally occurring conjugative plasmids containing bla(DHA-1) and bla(CMY-2) were obtained from K. pneumoniae isolates in western Sydney. These were moved into K. pneumoniae ATCC13883 and isogenic porin knockouts Kp885 (DeltaompK35) and Kp886 (DeltaompK36), created by homologous recombination of kanamycin resistance cartridges into the specified genes, and their antimicrobial susceptibilities compared. RESULTS: beta-lactam resistance was greater in the presence of CMY-2-containing plasmids than DHA-1-containing plasmids, and higher in K. pneumoniae than Escherichia coli. Neither cefepime nor imipenem resistance was observed, and DHA-mediated cefotaxime and ticarcillin/clavulanate resistance was unexpectedly reduced from 8-24 (CTX) and >256 (TIM) mg/L in Kp13883 to 1-2 (CTX) and 32-48 mg/L (TIM) in the isogenic DeltaompK36 porin knockout Kp886. CONCLUSIONS: AmpC plasmids in particular are an important cause of transmissible resistance to ticarcillin/clavulanate in K. pneumoniae, but probably not in E. coli. Single knockouts of OmpK35 and OmpK36 porins in K. pneumoniae do not significantly increase antibiotic resistance in K. pneumoniae, and a paradoxical lowering of resistance to CTX and TIM is seen with deletion of ompK36. This has potentially important clinical implications.
Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Microbiana/genética , Klebsiella pneumoniae/genética , Porinas/genética , beta-Lactamasas/genética , Técnicas de Inactivación de Genes , Klebsiella pneumoniae/enzimología , Pruebas de Sensibilidad Microbiana , Mutación , Plásmidos , Reacción en Cadena de la Polimerasa , Porinas/deficienciaRESUMEN
Diagnostic algorithms in commonly used automated bacterial identification systems fail to reliably identify a metallo-beta-lactamase in the Enterobacteriaceae. Misidentification as an extended-spectrum beta-lactamase may result in inappropriate dismissal of drugs such as aztreonam in favor of carbapenems, which may in turn select for a highly carabapenem resistant phenotype.