Carbapenemase-producing Enterobacteriaceae isolates collected in Portuguese hospitals

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

Polyclonal KPC-3-producing Enterobacteriaceae in Portugal

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

Human Extracellular-Matrix Functionalization of 3D hiPSC-Based Cardiac Tissues Improves Cardiomyocyte Maturation

The work here presented was funded by Fundacao para a Ciencia e Tecnologia (FCT) projects NETDIAMOND (SAICTPAC/0047/2015), financially supported by FEEI-Lisboa2020 and FCT/POCI-01-0145-FEDER-016385, and MetaCardio (PTDC/BTM-SAL/32566/2017); iNOVA4-Health -UIDB/04462/2020 and UIDP/04462/2020, a program financially supported by FCT/Ministerio da Ciencia, Tecnologia e Ensino Superior, through national funds is acknowledged; Funding from INTERFACE Programme, through the Innovation, Technology and Circular Economy Fund (FITEC), is gratefully acknowledged; and EU-funded projects BRAV3 (H2020, ID:874827) and ERAatUC (ref. 669088). HVA, AFL, and DS were financed by FCT Grants SFRH/BPD/120595/2016 and PD/BD/139078/2018 and PD/BD/106051/2015, respectively.Human induced pluripotent stem cells (hiPSC) possess significant therapeutic potential due to their high self-renewal capability and potential to differentiate into specialized cells such as cardiomyocytes. However, generated hiPSC-derived cardiomyocytes (hiPSC-CM) are still immature, with phenotypic and functional features resembling the fetal rather than their adult counterparts, which limits their application in cell-based therapies, in vitro cardiac disease modeling, and drug cardiotoxicity screening. Recent discoveries have demonstrated the potential of the extracellular matrix (ECM) as a critical regulator in development, homeostasis, and injury of the cardiac microenvironment. Within this context, this work aimed to assess the impact of human cardiac ECM in the phenotype and maturation features of hiPSC-CM. Human ECM was isolated from myocardium tissue through a physical decellularization approach. The cardiac tissue decellularization process reduced DNA content significantly while maintaining ECM composition in terms of sulfated glycosaminoglycans (s-GAG) and collagen content. These ECM particles were successfully incorporated in three-dimensional (3D) hiPSC-CM aggregates (CM+ECM) with no impact on viability and metabolic activity throughout 20 days in 3D culture conditions. Also, CM+ECM aggregates displayed organized and longer sarcomeres, with improved calcium handling when compared to hiPSC-CM aggregates. This study shows that human cardiac ECM functionalization of hiPSC-based cardiac tissues improves cardiomyocyte maturation. The knowledge generated herein provides essential insights to streamline the application of ECM in the development of hiPSC-based therapies targeting cardiac diseases.publishersversionpublishe

Epidemiology and zoonotic potential of Escherichia coli CTX-M-15-producing isolates in Portugal

Background: 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

Antimicrobial Susceptibility of Invasive Streptococcus pneumoniae Isolates in Portugal over an 11-Year Period

This national surveillance study presents the in vitro activities of the main antimicrobial agents against 1,331 S. pneumoniae isolates as tested by an agar dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (formerly NCCLS). The strains were isolated in several regions of Portugal from cases of invasive disease over an 11-year period (1994 to 2004). This study shows that the percentage of penicillin-nonsusceptible strains increased from 12% in 1994 to 28.5% in 2000. Then the rate declined to 17.7% in 2003 but increased again to 23.2% in 2004. Nevertheless, the rate of highly resistant isolates declined consistently, to 0.9% in 2001 to 2004. Ceftriaxone- and cefotaxime-nonsusceptible isolates became less frequent, from 4% and 8%, respectively, in 1994 to ≤1% in 2004. The macrolide-lincosamide-streptogramin B phenotype was the predominant macrolide phenotype found. The increase in the percentage of isolates that were only nonsusceptible to erythromycin (3.7% in 1994 to 1998 to 9.1% in 2002 to 2004) was similar to that for isolates with coresistance to penicillin and erythromycin (3.3% in 1994 to 1998 to 9.1% in 2002 to 2004). The nonsusceptibility to ciprofloxacin increased during recent years, from 0.5% in 2002 to 3.5% in 2004. Multidrug resistance also increased in recent years: from 7.9% in 2002 to 15.6% in 2004. The increasing use of macrolides could be causing the increase in penicillin and multidrug resistance, due to the coresistance to macrolides. The use of penicillin to treat empirical invasive pneumococci infections may need to be reconsidered

Phenotypic and molecular characterization of CMY-46 and CMY-50, two novel plasmid-mediated AmpC β-lactamase carried by Escherichia coli

Objectives: 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 Portugal

All 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.info:eu-repo/semantics/publishedVersio

Emergence of β-lactamase-mediated resistance to oxyimino-β-lactams in Enterobacteriaceae isolates in various services in a single centre

We 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