A simple, robust and rapid approach to detect carbapenemases in Gram-negative isolates by MALDI-TOF mass spectrometry: validation with triple quadripole tandem mass spectrometry, microarray and PCR.

Carbapenemases should be accurately and rapidly detected, given their possible epidemiological spread and their impact on treatment options. Here, we developed a simple, easy and rapid matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based assay to detect carbapenemases and compared this innovative test with four other diagnostic approaches on 47 clinical isolates. Tandem mass spectrometry (MS-MS) was also used to determine accurately the amount of antibiotic present in the supernatant after 1 h of incubation and both MALDI-TOF and MS-MS approaches exhibited a 100% sensitivity and a 100% specificity. By comparison, molecular genetic techniques (Check-MDR Carba PCR and Check-MDR CT103 microarray) showed a 90.5% sensitivity and a 100% specificity, as two strains of Aeromonas were not detected because their chromosomal carbapenemase is not targeted by probes used in both kits. Altogether, this innovative MALDI-TOF-based approach that uses a stable 10-μg disk of ertapenem was highly efficient in detecting carbapenemase, with a sensitivity higher than that of PCR and microarray.

Investigação de genes de resistência às quinolonas e aminoglicosídeos em Klebsiella pneumoniae produtoras de carbapenemases do tipo KPC em hospitais do estado de São Paulo

Pós-graduação em Microbiologia - IBILCE

Persistência de plasmídeos que codificam carbapenemases do tipo New-Delhi-Metalo-β-Lactamase

As metalo-β-lactamases (MBL) são capazes de hidrolisar os carbapenêmicos, a classe de antimicrobianos com maior potência para o tratamento de infecções graves e de maior uso clinico. Dentre as MBL, o grupo mais recentemente descrito e que apresentou rápida disseminação em todo o mundo é o da New-Delhi-Metalo- β-lactamases (NDM). Nas enterobactérias, os genes que codificam essas enzimas estão mais frequentemente localizados em plasmídeos. O estudo da estabilidade de plasmídeos que albergam o gene blaNDM-1 é importante para entender a predominância de espécies que carregam esses plasmídeos, desvendar mecanismos moleculares envolvidos na sua persistência e para desenvolver novas drogas que possam diminuir a sua persistência. Estudos recentes sobre estabilidade plasmidial evidenciaram que a maprotilina é capaz de induzir perda plasmidial de até 90% em E. coli K12. Neste trabalho, foi estudado o efeito da maprotilina na indução de cura de plasmídeos, que albergam o gene blaNDM-1, em diferentes espécies da família Enterobacteriaceae. Nove isolados pertencentes a diferentes espécies foram incluídas no estudo. Os plasmídeos foram caracterizados quanto ao seu tamanho por eletroforese e por sequenciamento de DNA no sistema Illumina. A persistência plasmidial foi determinada pelo método de contagem em placa em LB ágar com e sem tratamento com maprotilina em concentrações sub-inibitórias (50mg/L). O experimento foi conduzido por 10 dias, representando aproximadamente 100 gerações. Neste estudo evidenciou-se que o grupo das enterobactérias estão envolvidas na disseminação de plasmídeos com blaNDM-1, sendo que plasmídeos do grupo IncF estão mais relacionados a essa dispersão. A maprotilina teve efeito de cura plasmidial em todos os isolados exceto em E. hormaechei \"subsp. oharae\" e C. freundii. O isolado P. rettgeri apresentou maior taxa de perda plasmidial e a análise comparativa da sequência nucleotídica do plasmídeo indicou que a presença da IS5 pode estar relacionada com a diminuição da persistência plasmidial. Diferenças na persistência plasmidial, quando tratados com maprotilina, entre E. hormaechei \"subsp. steigerwaltii\" e E. hormaechei \"subsp. oharae\" sugerem que E. hormaechei \"subsp. oharae\" pode ser um possível disseminador de plasmídeos albergando blaNDM-1, devido a processos de adaptação co-evolutivos.

Enterobacteriaceae produtoras de carbapenemases : um novo desafio no controlo da infecção hospitalar

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Dissemination of bla(IMP-1)-carrying integron In86 among Klebsiella pneumoniae isolates harboring a new trimethoprim resistance gene dfr23

The genetic context of the bla(IMP-1) gene was evaluated in 9 Klebsiella pneumoniae isolates recovered from 2 hospitals in Sao Paulo, Brazil. All isolates harbored a copy of In86 carrying bla(Imp-1), aac(6`)-31, and aadAl. Eight strains from the same hospital also carried another class I integron harboring a new trimethoprim resistance gene (dfr23) that was chromosomally embedded. In86 was likely to be in a 30-kb nontransferable plasmid and was flanked upstream by a sequence identical to one identified in an IMP-1-producing Pseudomonas putida isolate. The bla(IMP-1)-carrying integron In86 was recently reported from nonfermentative bacilli isolated in Sao Paulo. These isolates appear to be the Source of this integron now acquired by K. pneumoniae strains from different hospitals in the same city. Metallo-beta-lactamase production is still rare among Enterobacteriaceae isolates in Brazil, but the acquisition of genetic structures carrying these mobile resistance determinants is worrisome and could lead to an increase in the prevalence of these phenotypes of resistance. (C) 2008 Elsevier Inc. All rights reserved.

Estudo dos principais mecanismos de resistência aos antibióticos β-lactâmicos em bactérias patogénicas de gram negativo

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Indications of carbapenem resistance evolution through heteroresistance as an intermediate stage in Acinetobacter baumannii after carbapenem administration

We describe an in vivo evolution of an antimicrobial profile from susceptibility to full-resistance to carbapenems, with heteroresistance as an intermediate stage, in an Acinetobacter baumannii strain. Heteroresistance was characterized by the growth of sub-populations within the susceptibility halo in both disk-diffusion and Etest. PCRs for the main A. baumannii carbapenemases were negative. The exact resistance mechanism, diagnostic methods and clinical relevance of heteroresistance in A. baumannii warrant further investigations. This is the first description of such phenomenon in vivo and the second report of heteroresistance to carbapenems in A. baumannii.

Ocorrência de cepas de Pseudomonas aeruginosa multirresistentes produtoras de metalo-β-lactamase blaSPM-1 em amostras clínicas

Este estudo avaliou a ocorrência de genes para metalo β-lactamases em isolados clínicos de Pseudomonas aeruginosa do Hospital São Vicente de Paulo, RS. Os genes foram pesquisados por PCR e o perfil de susceptibilidade foi avaliado por disco-difusão. Foram analisadas 46 cepas, sendo que cinco apresentaram o gene blaSPM-1.

Extending the reservoir of bla IMP-5: the emerging pathogen Acinetobacter bereziniae

Acinetobacter bereziniae clinical relevance is starting to be recognized; however, very few descriptions of its carbapenem resistance currently exist. Here we characterize two carbapenem-resistant A. bereziniae isolates. Materials & methods: Isolates were obtained from environmental and clinical samples. Carbapenemases were searched by phenotypic, biochemical and PCR assays. Clonality was studied by ApaI-PFGE and genetic location for carbapenemase genes were assessed by I-CeuI and S1 hybridizations. Results: Isolates were not clonally related but both produced the exclusively Portuguese IMP-5, with the clinical isolate also producing an OXA-58. The carbapenemase genes were plasmid located. Conclusion: Our results emphasize the role of non-baumannii Acinetobacter species as important reservoirs of clinically relevant resistance genes that could also contribute to their emergence as nosocomial pathogens

Changing the epidemiology of carbapenem-resistant Pseudomonas aeruginosa in a Brazilian teaching hospital: the replacement of São Paulo metallo-β-lactamase-producing isolates

In Brazil, carbapenem-resistant Pseudomonas aeruginosa isolates are closely related to the São Paulo metallo-β-lactamase (SPM) Brazilian clone. In this study, imipenem-resistant isolates were divided in two sets, 2002/2003 and 2008/2009, analysed by pulsed field gel electrophoresis and tested for the Ambler class B metallo-β-lactamase (MBL) genes blaSPM-1, blaIMP and blaVIM. The results show a prevalence of one clone related to the SPM Brazilian clone in 2002/2003. In 2008/2009, P. aeruginosa isolates were mostly MBL negative, genetically diverse and unrelated to those that had been detected earlier. These findings suggest that the resistance to carbapenems by these recent P. aeruginosa isolates was not due to the spread of MBL-positive SPM-related clones, as often observed in Brazilian hospitals.

Draft genome sequences of three NDM-1-producing Enterobacteriaceae species isolated from Brazil

The emergence of multidrug-resistant Enterobacteriaceae strains producing carbapenemases, such as NDM-1, has become a major public health issue due to a high dissemination capacity and limited treatment options. Here we describe the draft genome of three NDM-1-producing isolates: Providencia rettgeri(CCBH11880), Enterobacter hormaecheisubsp. oharae(CCBH10892) and Klebsiella pneumoniae(CCBH13327), isolated in Brazil. BesidesblaNDM-1, resistance genes to aminoglycosides [aadA1, aadA2,aac(6’)-Ib-cr] and quinolones (qnrA1,qnrB4) were observed which contributed to the multidrug resistance profile. The element ISAba125 was found associated to theblaNDM-1 gene in all strains.

Targeted therapy against multi-resistant bacteria in leukemic and hematopoietic stem cell transplant recipients: guidelines of the 4th European Conference on Infections in Leukemia (ECIL-4, 2011).

The detection of multi-resistant bacterial pathogens, particularly those to carbapenemases, in leukemic and stem cell transplant patients forces the use of old or non-conventional agents as the only remaining treatment options. These include colistin/polymyxin B, tigecycline, fosfomycin and various anti-gram-positive agents. Data on the use of these agents in leukemic patients are scanty, with only linezolid subjected to formal trials. The Expert Group of the 4(th) European Conference on Infections in Leukemia has developed guidelines for their use in these patient populations. Targeted therapy should be based on (i) in vitro susceptibility data, (ii) knowledge of the best treatment option against the particular species or phenotype of bacteria, (iii) pharmacokinetic/pharmacodynamic data, and (iv) careful assessment of the risk-benefit balance. For infections due to resistant Gram-negative bacteria, these agents should be preferably used in combination with other agents that remain active in vitro, because of suboptimal efficacy (e.g., tigecycline) and the risk of emergent resistance (e.g., fosfomycin). The paucity of new antibacterial drugs in the near future should lead us to limit the use of these drugs to situations where no alternative exists.

Blood culture-based diagnosis of bacteraemia: state of the art.

Blood culture remains the best approach to identify the incriminating microorganisms when a bloodstream infection is suspected, and to guarantee that the antimicrobial treatment is adequate. Major improvements have been made in the last years to increase the sensitivity and specificity and to reduce the time to identification of microorganisms recovered from blood cultures. Among other factors, the introduction in clinical microbiology laboratories of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology revolutionized the identification of microorganisms whereas the introduction of nucleic-acid-based methods, such as DNA hybridization or rapid PCR-based test, significantly reduce the time to results. Together with traditional antimicrobial susceptibility testing, new rapid methods for the detection of resistance mechanisms respond to major epidemiological concerns such as methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase or carbapenemases. This review presents and discusses the recent developments in microbial diagnosis of bloodstream infections based on blood cultures.

Unusual association of NDM-1 with KPC-2 and armA among Brazilian Enterobacteriaceae isolates

We report the microbiological characterization of four New Delhi metallo-β-lactamase-1 (blaNDM-1)-producing Enterobacteriaceae isolated in Rio de Janeiro, Brazil. blaNDM-1 was located on a conjugative plasmid and was associated with Klebsiella pneumoniae carbapenemase-2 (blaKPC-2) or aminoglycoside-resistance methylase (armA), a 16S rRNA methylase not previously reported in Brazil, in two distinct strains of Enterobacter cloacae. Our results suggested that the introduction of blaNDM-1 in Brazil has been accompanied by rapid spread, since our isolates showed no genetic relationship.

Tipificación y perfiles de resistencia antibiótica en unidades neonatales de Bogotá durante los años 2008-2012

La sepsis neonatal ha sido un problema en las unidades de cuidado intensivo al rededor del mundo. Su incidencia aumenta diez veces cuando el recién nacido es de muy bajo peso. El aislamiento de gérmenes resistentes en los cultivos tomados a los recién nacidos ha permitido identificar el comportamiento de los antibióticos de uso común. El presente estudio analítico de corte transversal realizado durante los años 2008 al 2012 en diez unidades de cuidados intensivos neonatales de la ciudad de Bogotá analizó la base de datos de los laboratorios de microbiología, obteniendo un total de 22.153 muestras de las cuales 7.132 ( 32.9%) fueron elegibles. Utilizando el software Whonet 5.6 y tomando solo el primer aislamiento por año y por paciente se realizaron análisis descriptivos y de resistencia bacteriana. Los resultados obtenidos fueron similares a los reportados en la literatura donde los gérmenes gran positivos son los más comúnmente aislados con un 53.2%, seguidos en importancia por los gérmenes gran negativos. Klebsiella pneumoniae y Escherichia coli en conjunto aportan 27.22%, presentando un aumento en el porcentaje de BLEE (Betalactamasa de Espectro Extendido) y aparición de carbapenemasas durante los años de estudio. El Acinetobacter baumannii ha duplicado la resistencia a ampicilina sulbactam llegando al 66.7%. Las candidas fueron aisladas en un porcentaje muy bajo sin documentarse resistencias. El Estafilococo aureus no presenta resistencias que sugiera la aparición de nuevos clones.