Browsing by Author "Louro, Deolinda"
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- Carbapenemase-producing Enterobacteriaceae isolates collected in Portuguese hospitalsPublication . Manageiro, Vera; Ferreira, Eugénia; Louro, Deolinda; Caniça, Manuela; Antibiotic Resistance Surveillance Program in Portugal (ARSIP)Objectives: In Portugal, little is known on carbapenemase (CARB)-producing Enterobacteriaceae. The aim of this study was to identify the resistance mechanisms of Enterobacteriaceae isolates, identified at hospital laboratories as carbapenem (CA) non-susceptible. Methods: This study included 61 Enterobacteriaceae isolates (26 Klebsiella spp, 15 Escherichia coli, 9 Enterobacter spp, 6 Morganella morgannii, 4 Proteus mirabilis, 1 Serratia marcescens), collected between 04/2006 and 09/2011 and sent to the NIH-Lisbon for CA susceptibility confirmation. Antimicrobial susceptibility of clinical isolates was performed by disk diffusion method (CA-SFM). Clinical isolates showing synergism between CA and boronic acid (BOR) (and/or clavulanic acid, CLAV) or with EDTA were considered presumptively CARB-producers from class A or Class B, respectively. PCR and sequencing were applied to detect and identify CARB-encoding genes; the respective genetic environment was revealed by sequencing using PCR mapping. Direct transfer of the CA resistance phenotype was attempted by mating-out assays. Antibiotics susceptibility (MIC) of transconjugants and respective isolates were tested by microdilution. Results: The majority of isolates were collected from the urine (57.4%) of elderly (≥65 years old) male patients (54.1%), admitted at the emergency room/ambulatory (24.6%) and at internal medicine (18.0%) wards. Among all isolates, 50.8% were nonsusceptible to at least one CA, being 67.2% multidrug-resistant; 16 isolates showed synergy between CA and BOR (and/or CLAV). Among those, 5 were KPC-3-producers (4 Klebsiella pneumoniae and 1 Enterobacter clocae), collected in 2010 (2) and 2011 (3). The blaKPC-3 genes were confirmed to be carried by plasmids. Genetic environment of blaKPC-3 gene revealed the presence of a Tn4401 transposon in all but one isolate (E. cloacae), suggesting that this last gene was included in other Tn4401-like isoform. We also detected a VIM-2-producing Klebsiella oxytoca, collected in 2009, among the 7 isolates that showed synergy between imipinem and EDTA. No blaGES, blaNDM or blaIMP were detected. Conclusion: In conclusion, this study provides new data regarding the molecular epidemiology of CARB-producing Enterobacteriaceae in Portugal. Overall, our results emphasize the need of a concerted action to manage CA use. This is supported by EARS-Net, which reported an increase in CA nonsusceptibility of K. pneumoniae isolates from 0.72% in 2008 to 1.58% in 2010.
- Disseminação de isolados de Enterobacteriaceae produtores da carbapenemase KPC-3Publication . Manageiro, Vera; Ferreira, Eugénia; Louro, Deolinda; Antimicrobial Resistance Surveillance Program in Portugal; Caniça, Manuela
- Diversity of extended-spectrum and plasmid-mediated AmpC β-lactamases in Enterobacteriaceae isolates from Portuguese health care facilitiesPublication . Jones-Dias, Daniela; Manageiro, Vera; Ferreira, Eugénia; Louro, Deolinda; Antibiotic Resistance Surveillance Program in Portugal (ARSIP) participants; Caniça, ManuelaA group of 124 Enterobacteriaceae isolates resistant to third generation cephalosporins, and collected in distinct health care facilities of different Portuguese regions was analysed. The great majority of the isolates were also resistant to fourth generation cephalosporins (83.9%), monobactam (96%), amoxicillin plus clavulanic acid (85.5%), and piperacillin plus tazobactam (66.9%). Overall, 84.7% (105/124) were multidrug resistant. Molecular methods enabled us to identify 86.3% (107/124) extended-spectrum β-lactamases (ESBL) producers, revealing a diversity of class A β-lactamases from different families, like TEM (TEM-1, TEM-10, TEM-24, and TEM-52), SHV (SHV-1, SHV-12, and SHV-28), CTX-M (CTX-M-1, CTX-M-9, CTX-M-14, CTX-M-15, and CTXM-32), and GES (GES-1). We have also detected class C enzymes like plasmid-mediated AmpC β-lactamases (PMAβs, DHA-1, and CMY-2) and chromosomal AmpCs in Enterobacter and Citrobacter spp. The PMAβ genetic context mapping suggests association with mobile elements, plasmid importation and the potential emergence of these β-lactamases. The most prevalent β-lactamase detected was CTX-M-15 (66.1%) and in 41.1% of the isolates it was associated with TEM-, OXA-type β-lactamases and Aac(6)᾿Ib-cr, which might indicate that the respective genotype has settled in our country. Indeed, CTX-M-15 was distributed amongst distinct clinical settings of several health care facilities (93.5%) from various regions. We provide evidence of a concerning clinical situation that includes vast occurrence of ESBLs, the settling of CTX-M β-lactamases, and the report of plasmidic and chromosomal AmpC in Portugal.
- Emergence of β-lactamase-mediated resistance to oxyimino-β-lactams in Enterobacteriaceae isolates in various services in a single centrePublication . Manageiro, Vera; Ferreira, Eugénia; Jones-Dias, Daniela; Louro, Deolinda; Pinto, Margarida; Diogo, José; Caniça, ManuelaWe studied 193 Enterobacteriaceae isolates presenting diminished susceptibility to oxyimino-cephalosporins recovered in a Portuguese hospital (2004–2008). CTX-M-3 producers, firstly detected in Portugal, were associated with a Klebsiella pneumoniae microepidemic clone. Production of CTX-M–type enzymes (CTX-M-1/-3/-9/-14/-15/-32), age ≥65 years, and nosocomial infection were risk factors for higher nonsusceptibility to oxyimino-β-lactams. CMY-2 and DHA-1 β-lactamases were only identified in 1% of isolates.
- Epidemiology and zoonotic potential of Escherichia coli CTX-M-15-producing isolates in PortugalPublication . Caniça, Manuela; Clemente, Lurdes; Jones-Dias, Daniela; Manageiro, Vera; Themudo, Patrícia; Albuquerque, Teresa; Francisco, Ana Patrícia; Louro, Deolinda; Ferreira, EugéniaBackground: The recent spread of plasmid-located CTX-M-ESBL-encoding genes is a serious threat to the clinical efficacy of expanded-spectrum cephalosporins. This study proposes to identify the epidemiology of plasmid-mediated CTX-M-encoding genes between an Escherichia coli strain isolated from a dolphin and several E. coli strains of human origin and, explain the responsible mechanism and reservoirs of current spread of CTX-M-type enzymes. Molecular typing of these strains establishes the linkage of dissemination between human and animal isolates. Methods: Sixty two ESBL-positive E. coli strains isolated from different clinical specimens in seven hospitals (2004 to 2009), from four different geographic regions, were screened for the presence of CTX-M encoding genes. An E. coli isolated from a respiratory exsudate in a dolphin in 2009, at the National Laboratory of Veterinary Research (LNIV) and characterized as CTX-M-producer, was also included in this study. Antimicrobial susceptibility was performed by broth-microdilution method. PCR and sequencing were used to screen and identify bla genes. Genetic relatedness among all isolates was examined by PFGE using XbaI enzyme. MLST was performed among the clinical epidemic human isolates clustering together and the isolate from the dolphin, according to the MLST database. Results: Forty eight human clinical isolates (77%) were CTX-M producers. Susceptibility towards beta-lactams confirmed all isolates as ESBL producers and also suggesting CTX-M enzymes expression. We detected blaCTX-M-15 (n=34), blaCTX-M-1 (n=4), blaCTX-M-3 (n=3), blaCTX-M-32 (n=3), blaCTX-M-14 (n=4), blaTEM-1 (n=39), and blaSHV-12 (n=8) genes; the dolphin isolate presented the blaCTX-M-15 gene. Genetic relatedness analysis by PFGE revealed one major cluster corresponding to a single epidemic clone A, which included 22 (35%) of all human isolates and the dolphin isolate, which clustered together with this clone A and, exhibited the same combination of MLST alleles across the seven sequenced loci, corresponding to the ST131. Twenty-one clinical isolates corresponding to the CTX-M-15-positive clone A were multidrug-resistant (95%), as well as the dolphin isolate. Conclusions: This study illustrated that genetic relatedness between human and animal E. coli CTX-M-15-producing clone strengthens its zoonotic potential. It may also explain the current spread of CTX-M-type enzymes worldwide among species from different origins and highlights the importance to identify antimicrobial resistance reservoirs, contributing to a single health for all.
- Genetic diversity and clonal evolution of carbapenem-resistant Acinetobacter baumannii isolates from Portugal and the dissemination of ST118Publication . Manageiro, Vera; Jones-Dias, Daniela; Ferreira, Eugénia; Louro, Deolinda; Antimicrobial Resistance Surveillance Program in Portugal (ARSIP); Caniça, ManuelaIn this study, 116 multidrug-resistant Acinetobacter baumannii (MDR-Ab) isolates recovered in various regions of Portugal were studied. All isolates were non-susceptible to tigecycline; one isolate was also non-susceptible to colistin, making it a step closer to pandrug resistance. Among 72 isolates tested by PFGE, 98.6% carried blaOXA-66, 1.4% blaOXA-104, 77.8% blaOXA-23, 23.6% blaOXA-24, 18.1% blaTEM-1 and 1.4% blaCTX-M-15-like genes. No OXA-58 or metallo-β-lactamase-encoding genes were detected. ISAba1 was found in 58/72 isolates (80.6%). Among these, ISAba1 was found upstream of blaOXA-51-like in 54 isolates. All but two of these isolates also carried ISAba1–blaOXA-23, highlighting the coexistence of ISAba1–blaOXA-51-like and ISAba1–blaOXA-23 genetic platforms, emphasising the importance of mobile genetic elements in the dissemination of carbapenem-hydrolysing class D β-lactamase genes. Tn2006-like and Tn2008-like, found within ST92 and ST118, may reflect either multiple genetic structures in the origin of blaOXA-23 acquisition or interclonal complex evolution. These results indicate that there may exist different genetic origins for carbapenem resistance among MDR-Ab isolates. Six PFGE profiles were associated with three major sequence types, with ST118 (OXA-23- or OXA-24-producer) being widely disseminated since 2009. ST98 (described so far as endemic in Portugal) and ST92 (which co-existed with ST98 before 2009) appeared to have been gradually replaced by ST118. The new ST188 (OXA-104-producer) was detected for the first time in this country. Identification of an extensively drug-resistant ST118 and carbapenem-resistant ST92, ST98 and ST118 isolates, both in community and healthcare facilities, demonstrates the menace of A. baumannii-associated infections.
- Phenotypic and molecular characterisation of carbapenem-hydrolysing class D beta-lactamase-producing Acinetobacter baumannii isolated in PortugalPublication . Manageiro, Vera; Jones-Dias, Daniela; Ferreira, Eugénia; Louro, Deolinda; Caniça, Manuela; the Antibiotic Resistance Surveillance Program in Portugal (ARSIP).Objectives: Acinetobacter baumannii is a ubiquitous pathogen able to cause colonization and both community and healthcare-associated infections. Our purpose was to evaluate the genetic relatedness of carbapenem-hydrolyzing class D b-lactamase (CHDL) producing MDR-Ab from different geographic regions of Portugal. The correlation with carbapenem resistance was also analysed. Methods: The study included 127 clinical A. baumannii isolates recovered in Portuguese hospitals (116 from Apr2009-Apr2010); 11 from 2005-2008 were included for genetic evolution comparison. The identification of strains was confirmed by PCR (based on the presence or absence of OXA-51-like genes) and antimicrobial susceptibility was screened by TSA and E-test. PCR and sequencing were applied to detect and identify genes encoding CHDLs, class B metallo-b-lactamases (MBL) (blaIMP and blaVIM), and class A b-lactamases (blaTEM, blaSHV and blaCTX-M). Genetic relatedness of MDR-Ab was examined by PFGE and MLST (typing of 25 isolates representative of different PFGE-clusters). Results: All isolates were MDR but susceptible to colistin. However, the MICs of colistin confirmed one isolate as nonsusceptible. Overall, 77% of the isolates carried blaOXA-23, 18% blaOXA-24, 28% blaTEM-1, 2% blaCTX-M-15-type and 1% blaTEM-110 genes. None of the isolates carried OXA-58 or MBL-encoding genes. All isolates carried the blaOXA-51-type gene, and expressed OXA-66 (98%), OXA-71 (1%) or OXA-104 (1%) CHDL. The blaOXA-104-CHDL gene carries 182 synonymous mutations comparing with the first-described b-lactamase. PFGE analysis revealed that 81 isolates clustered into six clones and 2 presented unique profiles. PFGE profiles were associated with five distinct STs, being ST118 the most frequently encountered, which was found in all 9 hospitals since 2009; this ST was followed by ST92, a single-locus variant (SLV) of ST118, which includes all but one isolate from 2009 and 2010. ST98, also a SLV of ST118, appeared since 2005. The ST187 appeared in 2006 and the novel ST188, appeared in 2009. Conclusion: This study provides updated data regarding the molecular epidemiology of MDR-Ab in Portugal. Here, we report the first appearance of two epidemic ST OXA-23-producers in the country, ST92 and ST118, suggesting clonal importation; during the study, both STs have successfully spread among all hospitals, suggesting clonal expansion. Indeed, they seem to be replacing the only ST described so far as endemic in Portugal (ST98).
- Phenotypic and molecular characterization of CMY-46 and CMY-50, two novel plasmid-mediated AmpC β-lactamase carried by Escherichia coliPublication . Manageiro, Vera; Louro, Deolinda; Ferreira, Eugénia; Caniça, ManuelaObjectives: The identification of isolates containing AmpC β-lactamases is epidemiologically and clinically relevant. With this study we performed the phenotypic and molecular characterization of two new CMY-2-types, designated CMY-46 and CMY-50, encountered among a total of 1664 clinical non-duplicate isolates of various Enterobacteriaceae species. Methods: E. coli INSRA1169 and INSRA3413 were isolated from the urine of patients with 77 years and 7 months old, hospitalized in the ward and in pediatrics, respectively. The blaCMY genes were cloned in the plasmid pBK-CMV and transformed into electrocompetent E. coli DH5α ∆ampC by electroporation. Antimicrobial susceptibility (MIC) was determined by a microdilution method. E. coli INSRA6015, a CMY-2-producer, was used for phenotype comparison. PCR-mapping of the genetic environment of new blaCMY genes was performed using primers for known antibiotic and mercury resistance genes. Results: Antimicrobial susceptibly tests showed that all isolates and respective transformants were nonsusceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime. INSRA1169 and INSRA6015 were also nonsusceptible to ciprofloxacin and to trimethoprim. Regarding gentamycin, only INSRA1169 was resistant. Its noteworthy that the transformants EcDH5a(pBK-CMY-2) and EcDH5a(pBK-CMY-46) exhibited higher values for extended-spectrum cephalosporins than the respective isolates. All strains were susceptible to cefepime and imipenem, showing synergy between cloxacilin and cefoxitin and/or ceftazidime. No phenotypic alterations were found comparing the new CMY-type with the parental CMY-2. The genetic characterization of CMY-46 and CMY-50-encoding genes revealed a Citrobacter freundii chromosome-type structure, encompassing a blc-sugE-blaCMY-2-type-ampR platform in both isolates. In addition, a sul1-type class 1 integron and a truncated mercury resistance operon were encountered. Conclusion: Although the CMY-type enzymes studied conferred resistance to extended-spectrum cephalosporins, the susceptibility to cefepime lead us to assume that those enzymes are not extended-spectrum cephalosporinases. Otherwise, the presence of three genetic resistance-encoding regions is of great concern, namely the truncated mercury resistance operon, which may help to promote antibiotic resistance through indirect selection.
- Polyclonal KPC-3-producing Enterobacteriaceae in PortugalPublication . Manageiro, Vera; Ferreira, Eugénia; Louro, Deolinda; Caniça, Manuela; Antimicrobial Resistance Surveillance Program in Portugal (ARSIP)Water has been recognized as a reservoir for antibiotic resistance genes (ARG), where the presence of mobile genetic elements, including plasmids, favors their dissemination. It is noteworthy that nonpathogenic environmental organisms, where plasmids encoding multiple ARG are prevalent, can provide resistance to most classes of antimicrobials including beta-lactams, aminoglycosides, chloramphenicol, trimethoprim, streptomycin, fosfomycin, quinolones, among others. The main goal of this study was to evaluate the presence of ARGs, related with -lactam and quinolone resistance, in Gram-negative bacteria isolates from surface and raw and treated waste water environments. Water samples were collected from different environments within an urban water cycle in the region of Northern Portugal, which included treated and raw wastewater, water to the consumers and water surface. Screening of antimicrobial susceptibility of 56 Gram-negative isolates (20 Escherichia coli, 8 Citrobacter spp., 7 Klebsiella spp., 6 Kluyvera spp., 4 Sphingomonas panni, 2 Enterobacter spp., 1 Acinetobacter johnsonii, 3 Aeromonas veronii, 1 Hafnia alvei, 1 Pantoea agglomerans, 1 Roultella ornithinolytica, 1 Serratia sp., 1 Stenotrophomonas maltophilia), identified by 16S rRNA gene sequencing analysis using universal primers, was performed by disk diffusion method. Interpretative reading of susceptibilities allowed to direct the search for antibiotic resistant genes. PCR and sequencing were used to screen and identify beta-lactamase- and plasmidmediated quinolone resistance (PMQRs)-encoding genes. All isolates were also screened for the presence of class 1 integrons. PCR-based replicon typing (PBRT) was used to type the resistance plasmids of the blaGES-5- producing isolate among the major incompatibility (Inc) groups, specifically FIA, FIB, FIC, HI1, HI2, I1-I , L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA. Multilocus sequence typing (MLST) of the GES-5 K. pneumoniae-producing isolate was performed according to the Institute Pasteur scheme (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html). Overall, 16/56 isolates were multidrug-resistant (MDR), i.e. presenting a reduced susceptibility to 3 or more structurally unrelated antibiotics, suggesting a great diversity of resistance mechanisms. Noteworthy, 10 isolates (4 S. panni, 1 A. johnsonii, 3 A. veronii, 1 K. pneumoniae, and 1 S. maltophilia) showed nonsusceptibility to carbapenems, which constitutes one of the last resorts on the antimicrobial therapy. Their phenotypic and molecular characterization revealed the expression of several enzymes: the naturally occurring carbapenemase in one S. maltophilia, ImiS in three A. veronii, both MBLs, and OXA-type carbapenemase in one A. johnsonii, responsible for their intrinsic resistance; the class A GES-5-producing K. pneumoniae isolate belonged to a novel MLST sequence type, the ST961 (18-22-18-90-142-13-179). PBRT of the plasmid-carrying blaGES-5 gene showed that it did not belong to any of the Inc groups tested. No carbapenemases were found in the 4 S. panni isolates. The beta-lactam resistance, carbapenem susceptibility, found in 33 isolates was justified by the presence of various Class A (12 blaTEM-1 with distinct promoters, 6 blaSHV) and different Class C -lactamase-encoding genes (blaCMY, blaACC, blaACT), some here firstly described: blaCMY-65 (JF780936), blaCMY-89 (HE819403), blaCMY-90 (HE819404), blaACT-13 (HE819402) and blaACC-5 (HE819401). Class 1 integrons were detected among 6 of TEM- 1-producing isolates. Together, the beta-lactamases identified explain the level of beta-lactam resistance. Besides quinolone resistance detected, none PMQR were identified, suggesting chromosomal alterations in the quinolone resistance-determining region. This study identified ARGs related not only to commonly used antibiotics, but also to carbapenems, providing, at our knowledge, the first description of a GES-5-producing Enterobacteriaceae recovered in an environmental setting. The study highlights the need of surveillance of these antibiotic resistance mechanisms in environmental backgrounds, since it represents a liable reservoir of potential pathogenic resistant bacteria. Worryingly, recent studies demonstrated that while the WWTP reduced the bacterial load, the treatment is inefficient to remove antibiotic resistant bacteria.
- Polyclonal KPC-3-producing Enterobacteriaceae in PortugalPublication . Manageiro, Vera; Ferreira, Eugénia; Louro, Deolinda; The Antimicrobial Resistance Surveillance Program in Portugal; Caniça, ManuelaAll 6 KPC-3-producing Enterobacteriaceae isolates (5 K. pneumoniae and 1 Enterobacter cloacae), identified among 61 isolates, from different Portuguese health institutions (March 2010 to December 2011) were multidrug resistant, showing susceptibility only to colistin. The blaKPC-3 gene, conferring resistance to carbapenemes, was present alone or in combination with other bla genes: blaSHV-26, blaCTX-M-15, and the ESBL blaSHV-164, here firstly described. The Tn4401-harbouring blaKPC-3 encountered in all isolates, showed a 68 bp deletion upstream of the bla gene. All but two KPC-3-containing plasmids revealed the following types: IncFrepB plus IncFIIs (n=3) and IncFrepB plus IncP (n=1). Dissemination of blaKPC seems to be due to carriage of similar KPC-harbouring plasmids within genetically distinct K. pneumoniae (ST14, ST34, ST59, ST416 and the novel ST960, by MLST) and E. cloacae clinical strains.