Browsing by Author "Barbosa, Stephanie"
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- Carbapenemase-producing multidrug resistant Enterobacteriaceae in Portugal: class A and BPublication . Caniça, Manuela; Manageiro, Vera; Almeida, Joana; Barbosa, Stephanie; Simões, Constança; Ferreira, EugéniaIntroduction: Carbapenems are often the antimicrobials of last resort to treat infections due to extended-spectrum β-lactamase (ESBL)- or plasmid-mediated AmpC (PMAβ)-producing Enterobacteriaceae isolates. Unfortunately, carbapenem non-susceptible Enterobacteriaceae have been reported worldwide mainly, because of the acquisition of carbapenemase-encoding genes. A large variety of carbapenemases has increasingly been identified in each of the four Ambler molecular classes. Thus, the main aim of this study was to search and characterize carbapenemase-producing Enterobacteriaceae (CPE) isolates recovered from Portuguese health care institutions. Methods: This study included 165 clinical isolates of Enterobacteriaceae nonsusceptible to ertapenem, identified among 2105 isolates, all recovered from different Portuguese healthcare institutions, between 2006 and 2013 that were sent to the NIH for antibiotic susceptibility confirmation in the context of its reference function. Screening of antimicrobial susceptibility was performed by disc diffusion method (www.sfm-microbiologie.org/). Clinical isolates with resistance or with decreased susceptibility to ertapenem were considered putative carbapenemase producers. For all CPE isolates and respective transconjugants, MICs of selected antibiotics were determined by the microdilution broth method (http://www.eucast.org/clinical_breakpoints/). PCR and sequencing were applied to detect and identify the main CPE-encoding genes from class A, B and/or D. The presence of blaESBL, blaPMAβ, and PMQR-encoding genes, were also investigated. Transferability of the carbapenemase genes was attended by broth mating-out assays. The plasmids obtained from transconjugants were characterized by PCR-based replicon typing. Genetic diversity of K. pneumoniae isolates was investigated by multilocus sequence typing (MLST), using the protocol developed by the Institute Pasteur (www.pasteur.fr/mlst/Kpneumoniae.html). Results: Thirty-five (21.2%) of the 165 positive isolates were confirmed to be CPE, of which the majority were collected from the urine (54.3%) of elderly (≥65 years old) male patients (54.3%), and admitted at the emergency room/ambulatory (22.9%) or internal medicine (17.1%) wards. All were multidrug-resistant with nonsusceptibility to at least one carbapenem; colistin was the only antibiotic effective against all CPE isolates. Tigecycline and ciprofloxacin were effective against 57.1% and 31.4%, respectively, of carbapenemase-producing isolates. We identify 30 blaKPC-3, 4 blaGES-5 and one blaVIM-2, alone or in combination with other bla and/or PMQR-encoding genes. MLST of the 30 K. pneumoniae isolates showed an important diversity, belonging to distinct STs. Conclusion: This study provides new data regarding the widespread of CPE in Portugal, which encoding genes are carried by similar plasmids. Overall, our results emphasize the need of a concerted action to manage carbapenem use.
- Molecular survey of carbapenem resistant Enterobacteriaceae isolates from Portuguese Hospitals: co-production of carbapenemase KPC-3 and the efflux pump OqxABPublication . Manageiro, Vera; Ferreira, Eugénia; Almeida, Joana; Barbosa, Stephanie; Simões, Constança; Antimicrobial Resistance Surveillance Program in Portugal (ARSIP); Caniça, ManuelaObjectives: Although there are important studies regarding the different carbapenemase (CARB)-producing Gram-negative bacteria, little is known concerning their molecular epidemiology in Portugal. The main aim of this study was to characterize, by phenotype and molecular typing methods, CARB-producing Enterobacteriaceae isolates recovered from Portuguese health care institutions, and evaluate its impact on treatment strategy. Methods: This study included 2105 clinical isolates, collected between April/2006 and February/2013, in different Portuguese healthcare institutions. Screening of antimicrobial susceptibility was performed by disc diffusion method. Clinical isolates with resistance or with decreased susceptibility to ertapenem were considered presumptively CARB-producers; in these isolates, PCR and sequencing were applied to detect and identify CARB-encoding genes, as well as other bla and plasmid-mediated quinolone resistance (PMQRs) genes. MICs of CARB-producing isolates were tested by microdilution (EUCAST breakpoints). The plasmids obtained from clinical isolates were characterized by PCR-based replicon typing (PBRT). Clonal relatedness of K. pneumoniae isolates was investigated by multilocus sequence typing (MLST), using the protocol developed by the Institute Pasteur (www.pasteur.fr/mlst/Kpneumoniae.html). Results: Among the 2105 isolates tested, 165 (7.8%) were putative CARB-producers and were selected for further analysis. Thirty-five (21.2%) of the 165 positive isolates were confirmed to be CARB-producers, of which the majority were collected from the urine (54.3%) of elderly (≥65 years old) male patients (54.3%), and admitted at the emergency room/ambulatory (22.9%) or internal medicine (17.1%) wards. All were multidrug-resistant, with nonsusceptibility to at least one carbapenem, and with consistent susceptibility only to colistin. In those isolates was detected the following beta-lactamases: 30 KPC-3 (22 K. pneumoniae, 3 Escherichia coli, 2 Enterobacter aerogenes and 3 Enterobacter clocae), 4 GES-5 (K. pneumoniae) and one VIM-2 (Klebsiella oxytoca). CARB-encoding genes were present alone or in combination with other bla genes, such as blaSHV-12, blaSHV-14, blaSHV-26, blaSHV-36, blaCTX-M-15, and the blaSHV-164. PMQR-encoding genes were also detected, namely qnrA, qnrB, aac(6’)-Ib-cr and the recently identified oqxAB. All blaKPC-3 genes were located on a Tn3-based transposon, Tn4401, while blaGES-5 and blaVIM-2 genes were associated with class 3 and 1 integrons, respectively. In our study, the majority of the blaCARB-harbouring plasmids were nonconjugative, having been typed as IncFrepB by PBRT. Clonal relatedness of the 26 K. pneumoniae isolates, obtained by MLST, showed that they were from distinct STs, namely ST14, ST15, ST34, ST59, ST147, ST416, ST698, and from the two novels ST: ST960 and, among all, the predominant ST1138 (corresponding to KPC-3 plus SHV-36 producers). Conclusion: In conclusion, this study provides new data regarding the molecular epidemiology of CARB-producing Enterobacteriaceae, which appears to be widespread in Portugal. Dissemination of blaCARB seems to be due to carriage of similar CARB-harbouring plasmids within genetically diverse clinical strains. Overall, our results emphasize the need of a concerted action to manage carbapenem use.
- Predominance of KPC-3 in a Survey for Carbapenemase-Producing Enterobacteriaceae in PortugalPublication . Manageiro, Vera; Ferreira, Eugénia; Almeida, Joana; Barbosa, Stephanie; Simões, Constança; Bonomo, Robert A.; Caniça, ManuelaAmong the 2,105 Enterobacteriaceae tested in a survey done in Portugal, 165 were nonsusceptible to carbapenems, from which 35 (26 Klebsiella pneumoniae, 3 Escherichia coli, 2 Enterobacter aerogenes, and 3 Enterobacter cloacae isolates and 1 Klebsiella oxytoca isolate) were confirmed to be carbapenemase producers by the presence of 30 Tn4401d-blaKPC-3, 4 intI3-blaGES-5, and one intI1-blaVIM-2 gene, alone or in combination with other bla genes. The dissemination of blaKPC-3 gene carried by an IncF plasmid suggests lateral gene transfer as a major mechanism of dissemination.
- β-lactamase-producing Gram negative isolates and molecular mechanisms involved in the expression of tigecycline resistancePublication . Barbosa, Stephanie; Ferreira, Eugénia; Manageiro, Vera; Jones-Dias, Daniela; Antimicrobial Resistance Surveillance Program in Portugal (ARSIP); Caniça, ManuelaObjectives: The emergence of antibiotic resistant Gram negative bacteria, particularly β-lactamase-producing bacteria, seriously compromises the efficacy of the treatment options available. This study aimed to evaluate the antimicrobial susceptibility, and to characterize the tigecycline resistance due to efflux pump production, in Gram negative clinical isolates. Moreover, it meant to clarify the in vitro activity of tigecycline against isolates resistant to other antibiotic classes, such as β-lactams, eventually related to β-lactamase production. Material and Methods: A total of 211 isolates collected in 2010 and 2012, were studied. The antimicrobial susceptibility of all isolates was performed by disc diffusion method and minimal inhibitory concentration (MIC), and interpreted by the SFM guidelines. The antibiotics tested were the following: colistin, ciprofloxacin, imipinem, cefoxitin, ceftazidime, cefotaxime, cefotaxime, and cefotaxime/clavulanate, tigecycline (by MIC); trimethoprim/sulfamethoxazole, fosfomycin, nitrofurans, and other antibiotics from the beta-lactam class (namely carbapenemes), fluoroquinolones and aminoglycosides (by disc diffusion method). Beta-lactamase characterization was performed by isoelectric focusing, followed by PCR and sequencing of bla genes from Ambler class A (blaTEM, blaSHV, blaOXA, blaCTX-M, blaGES, blaKPC, blaSME, blaNMC), class C (blaMOX, blaCIT, blaDHA, blaACC, blaMIR, blaFOX, blaACT), and class D (blaOXA-48). The ramR (Klebsiella pneumoniae) and marR (Escherichia coli) efflux pump genes involved in tigecycline resistance were also investigated by PCR and sequencing. Positive controls were used in each method performed. Results: Susceptibility testing evaluation identified 73% of tigecycline susceptible isolates, and 89% of multidrug resistance, among which 70% were susceptible to this antibiotic. Although there was a high prevalence of β-lactam resistance, carbapenems were still effective against the majority of the isolates studied, as well as colistin and amikacin. Globally, molecular characterization allowed the detection of penicillinases, ESBLs from families CTX-M [CTX-M-15-(type), CTX-M-1, CTX-M-32 and CTX-M-14], TEM (TEM-4, TEM-10), SHV (SHV-2, SHV-12, SHV-55) and GES (GES-7), as well as carbapenemases (KPC-3) and AmpC β-lactamases (CMY-2, DHA-1, MIR-type); among those, 75% were susceptible to tigecycline. The molecular analyses of tigecycline resistance mechanisms revealed one deletion, one insertion and one to four point mutations in the ramR gene that might contribute to the overexpression of AcrAB efflux pump, in 9 out of 20 Klebsiella pneumoniae isolates showing reduced susceptibility. Considering the analyses of the marR gene from 12 Escherichia coli isolates (5 with and 6 without tigecycline resistance), two point mutations were detected. Conclusions: This study showed a great diversity of β-lactamases, such as ESBL, and the presence of carbapenemases in Gram negative bacteria. Nevertheless, the studied isolates still showed decisive susceptibility patterns to important antibiotic classes, namely tigecycline, carbapenems, colistin and amikacin. The modifications identified in genes regulating the AcrAB efflux pump are accountable for the tigecycline resistance, but also to several antimicrobial classes, contributing to the high multidrug resistance scenario.