Browsing by Author "Antimicrobial Resistance Surveillance Program in Portugal (ARSIP)"
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- 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.
- 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.
- 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.
- 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.