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 TecnologiaA resistência aos antibióticos é um problema global e emergente, sendo as β-lactamases o principal mecanismo de resistência bacteriana aos antibióticos β-lactâmicos em bactérias de Gram negativo. O principal objectivo deste trabalho consistiu em avaliar a diversidade das β-lactamases produzidas por um grupo de 126 bactérias de Gram negativo,isoladas no período de 2004 a 2008 em diversos ambientes clínicos, em particular as β-lactamases de espectro alargado (ESBL), AmpC e carbapenemases. O teste de susceptibilidade aos antibióticos, permitiu determinar a expressão fenotípica das β-lactamases produzidas pelos isolados em estudo e dirigiu ainda a pesquisa molecular dos genes que codificam essas enzimas, os genes bla, de acordo com esse fenótipo. As β-lactamases ESBL, identificadas por sequenciação nucleotídica daqueles genes e análise bioinformática, eram da família TEM (TEM-10, TEM-24 e TEM-52), SHV (SHV-12), CTXM (CTX-M-1, CTX-M-9, CTX-M-14, CTX-M-15 e CTX-M-32) e GES-1, sendo CTX-M-15 as enzimas mais comuns. Entre o total de isolados estudados 81 eram produtores desta enzima CTX-M-15, encontrando-se disseminada entre as várias espécies, provenientes de várias regiões do país o que, associado a dados anteriores, sugere a sua disseminação crescente,eventualmente clonal, nomeadamente a nível hospitalar. Aí verificou-se existir disseminada por vários serviços. Entre as β-lactamases de classe A de Ambler, foram ainda detectadas as penicilinases SHV-1, TEM-1 e SHV-28, estando relacionadas com falsos fenótipos ESBL identificados, devido a provável hiperprodução. A caracterização bioquímica de β-lactamases AmpC cromossómicas, realizada por focagem isoeléctrica, permitiu confirmar a expressão destas enzimas, tendo sido verificado que as estirpes de Pseudomonas, Citrobacter e Enterobacter spp co-produziam ainda β- lactamases CTX-M-14, CTX-M-15, SHV e TEM-tipo, focalizando em pIs pré-definidos. Entre as β-lactamases de classe C, foram ainda detectadas as AmpC plasmídicas DHA-1 e CMY-2, nunca antes descritas em Portugal. A avaliação dos parâmetros cinéticos para a nova enzima, SHV-99, revelou uma maior afinidade (Km) para as penicilinas, cefalosporinas de terceira geração e aztreonam, o que nos leva a sugerir que substituições aminoacídicas na posição 104 de Ambler podem estar envolvidas no reconhecimento e discriminação dos substratos.Em conclusão, a co-expressão frequente de duas ou mais β-lactamases nos isolados deste estudo, a diversidade de famílias de β-lactamases identificadas, nomeadamente em diferentes espécies, e a resistência a diferentes antibióticos β-lactâmicos que lhes está associada, partilhando sobretudo fenótipos de multirresistência, constitui um panorama preocupante que interessa valorizar em termos de saúde públic

Genetic Background and Expression of the New qepA4 Gene Variant Recovered in Clinical TEM-1- and CMY-2-Producing Escherichia coli

A new QepA4 variant was detected in an O86:H28 ST156-fimH38 Escherichia coli, showing a multidrug-resistance phenotype. PAβN inhibition ofqepA4-harboring transconjugant resulted in increase of nalidixic acid accumulation. TheqepA4andcatA1genes were clustered in a 26.0-kp contig matching an IncF-type plasmid, and containing a Tn21-type transposon with multiple mobile genetic elements. This QepA variant is worrisome because these determinants might facilitate the selection of higher-level resistance mutants, playing a role in the development of resistance, and/or confer higher-level resistance to fluoroquinolones in association with chromosomal mutations.VM was supported by FCT fellowship (grant SFRH/BPD/77486/2011), financed by the European Social Funds (COMPETE-FEDER) and national funds of the Portuguese Ministry of Education and Science (POPH-QREN). The authors thank Fundação para a Ciência e a Tecnologia (FCT) for project grant PEst-OE/AGR/UI0211/2011-2014, Strategic Project UI211-2011-2014info:eu-repo/semantics/publishedVersio

Assessing the antibiotic susceptibility of freshwater Cyanobacteria spp.

Freshwater is a vehicle for the emergence and dissemination of Antibiotic resistance. Cyanobacteria are ubiquitous in freshwater, where they are exposed to antibiotics and resistant organisms, but their role on water resistome was never evaluated. Data concerning the effects of antibiotics on cyanobacteria, obtained by distinct methodologies, is often contradictory. This emphasizes the importance of developing procedures to understand the trends of antibiotic susceptibility in cyanobacteria. In this study we aimed to evaluate the susceptibility of four cyanobacterial isolates from different genera (Microcystis aeruginosa, Aphanizomenon gracile, Chrisosporum bergii, Planktothix agradhii), and among them nine isolates from the same specie (M.aeruginosa) to distinct antibiotics (amoxicillin, ceftazidime, ceftriaxone, kanamycine, gentamicine, tetracycline, trimethoprim, nalidixicacid, norfloxacin). We used a method adapted from the bacteria standard broth microdilution. Cyanobacteria were exposed to serial dilution of each antibiotic (0.0015–1.6mg/L) in Z8 medium (20±1◦C; 14/10hL/D cycle; light intensity 16±421μEm−s−). Cell growth was followed overtime (OD450nm/microscopic examination) and the minimum inhibitory concentrations (MICs) were calculated for each antibiotic/ isolate. We found that β-lactams exhibited the lower MICs, aminoglycosides, tetracycline and norfloxacine presented intermediate MICs; none of the isolates were susceptible to trimethoprim and nalidixic acid. The reduced susceptibility of all tested cyanobacteria to some antibiotics suggests that they might be naturally non-susceptible to these compounds, or that they might became non-susceptible due to antibiotic contamination pressure, or to the transfer of genes from resistant bacteria present in the environment.ED, VM, and DJ have received research funding from Fundação para a Ciência e Tecnologia via grants SFRH/BPD/77981/2011, SFRH/BPD/77486/2011, and SFRH/BD/80001/2011, respectively. The authors thank Fundação para a Ciência e a Tecnologia (FCT) for project grant PEst-OE/AGR/UI0211/2011-2014, Strategic Project UI211-2011-2014

Accessing the molecular basis of transferable quinolone resistance in Escherichia coli and Salmonella spp from food-producing animals and products

Background: Salmonella and Escherichia coli resistant to quinolones frequently arise in animals, being easily transferred to humans through the food chain, which can ultimately lead to the development of untreatable infectious diseases. The aim of the present study was to investigate the presence of PMQR determinants among Salmonella spp and E. coli from food-producing animals and derivative food products. Methods: Salmonella spp (n=183) and E. coli (n=182) isolates were collected from food-producing animals (n=274) and derivative food products (n=91). Antimicrobial susceptibility testing was performed by standard disk diffusion method, according to the CA-SFM veterinary guidelines. PCR and sequencing were used to detect PMQR- (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6’)-Ib-cr, and qepA) and β-lactamase-encoding genes (blaTEM, blaSHV, blaOXA and ampC) and to examine the QRDR of gyrA, gyrB, parC and parE genes in PMQR positive isolates. Plasmid characterization was accessed by conjugation followed by replicon-typing. Genetic relatedness of PMQR positive E. coli was examined by MLST and Salmonella isolates were serotyped according to the Kauffmann-White scheme. Mobile genetic elements were also investigated through PCR mapping assays. Results: Overall, 4.7% (17/365) harbored Qnr-encoding genes from qrnB and qnrS families, specifically qnrB2 (n=3), qrnB19 (n=3), and qnrS1 (n=11). All but one isolate presented at least one mutation in QRDR region of genes gyrA, parC or parE genes. 35.3% of Qnr-producing isolates presented resistance to β-lactam antibiotics that were justified by the presence of β-lactamases from TEM (TEM-1, n=10; and TEM-135, n=1) and SHV (SHV-108, n=1) families in QnrB19- and QnrS1-harbouring isolates. All but one Qnr-producing isolates were positively typed by replicon-typing, varying among IncN (n=2), IncFIB (n=11), IncFIC (n=3), IncI1 (n=2), IncHI2 (n=5), IncY (n=1) and IncL/M (n=3) and were, mostly, genetic unrelated. Qnr genes were detected nearby several mobile elements like ISEcl2, IS26 and ISCR1. Conclusions: This study illustrated the existence of Qnr-producing E. coli and Salmonella from food-producing animals, associated to specific mobile elements that can mediate their transference between species and among distinct settings. Epidemiology of PMQR mechanisms and the dissemination of plasmids carrying Qnr-encoding genes in veterinary isolates can compromise the efficacy of fluroquinolone treatments in both animals and humans

Draft Genome Sequence of a Pathogenic O86:H25 Sequence Type 57 Escherichia coli Strain Isolated from Poultry and Carrying 12 Acquired Antibiotic Resistance Genes

Free PMC Article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582591/Escherichia coli is a commensal bacterium that is frequently associated with multidrug-resistant zoonotic and foodborne infections. Here, we report the 5.6-Mbp draft genome sequence of an E. coli recovered from poultry, which encodes multiple acquired antibiotic resistance determinants, virulence factors, pathogenicity determinants, and mobile genetic elements.Daniela Jones-Dias and Vera Manageiro have received research funding from Fundação para Ciência e Tecnologia (grant numbers SFRH/BD/ 80001/2011 and SFRH/BPD/77486/2011, respectively). This work was supported by Fundação para a Ciência e a Tecnologia (grant number PEst-OE/AGR/UI0211/2011-2014)

Multilocus Sequence Typing of Vancomycin-Resistant Enterococcus faecium isolated from pigs in Portugal

Vancomycin-resistant enterococci (VRE) first appeared in the late 1980s in a few European countries. In the last two decades, however, vancomycin-resistant Enterococcus faecium (VREfm) as became an emergent and challenging nosocomial problem. Specific clonal groups of E. faecium show an enhanced capacity to disseminate in the nosocomial setting. These strains can be assigned to distinct clonal groups or complexes based on DNA sequence-based typing (multi-locus sequence typing - MLST). In this context, we used the MLST technic to study the clonal relatedness of 18 VREfm strains previously isolated from pigs at slaughter level, in Portugal. These strains have been phenotypic and genotypic characterized in a previous study (1). For this purpose, internal 400–600-bp fragments of housekeeping genes were amplified and sequenced: adk, atpA, ddl, gdh, gyd, purK and pst (2). The sequences obtained were analysed and compared against the http://mlst.ucc.ie/ database. The combination of the seven obtained alleles, for each isolate, allows us to determine the corresponding sequence type (ST) and clonal complex (CC). MLST analyses revealed sequence type 5 (ST5) (n =5) and ST139 (n=12). These E. faecium sequence types belong to clonal complex 5 (CC5). Although ST139 is farthest from ST5, from which differs in three alleles, it also belongs to CC5. Strains belonging to CC5 are recognized to be circulating among European pigs. Although E. faecium CC5 are commonly found among animals they have also been isolated from humans. Furthermore, four of the isolates assigned to ST5 showed high-level resistance (HLR) to kanamycin and streptomycin, what can be of concern. In case of severe enterococcal infections the synergistic and bactericidal therapy can be reliably achieved with the addition of an aminoglycoside to β-lactamic antibiotics (or other cell wall agent such as vancomycin), as long as the organism does not exhibit HLR to the aminoglycoside. The recovery of E. faecium CC5 clone from slaughtered animals is of concern, since these strains may have the ability to either colonize humans or cause human infections

Multilocus Sequence Typing for characterization of potential risk ESBLs-producing Escherichia coli isolated from pigs, including strains of new singletons ST2528, ST2524 and ST2525

Infections caused by Escherichia coli harboring extended-spectrum beta-lactamases (ESBL) have a tremendous impact on public health, because of treatment complications. ESBL-producing E. coli are increasingly reported in healthy food-producing animals that can spread to humans either by direct contact or, more importantly, through the food chain. Here we describe a molecular survey aimed at determining the population structure and dynamics of ESBL-producing E. coli strains recovered from healthy pigs slaughtered for human consumption in Portugal. For this purpose, a total of 71 faecal samples from pigs were collected (2008 to 2009) in different geographical regions of Portugal. Susceptibility to 16 antibiotics was tested by disk-diffusion method in all recovered isolates and ESBL detection was carried out by double-disk test. PCR and sequencing methods characterized blaESBL genes responsible for the ESBL-phenotype. In addition, we used multilocus sequence typing (MLST) to identify the genetic lineages of all ESBL-producing E. coli strains, which were characterized by sequencing the internal fragments of 7 housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, recA); the MLST database was used to determine allelic profiles and for sequence type (ST) and clonal complex (CC) assignment. Among the 35 ESBL-producing strains, MLST analysis revealed 9 different STs under 6 CCs and 9 singletons STs. The CC10 and CC155 were the most common CCs, with 4 and 11 isolates, respectively. Two other isolates were assigned to the CC101. Moreover, 5 strains were included in 3 new STs; 3 of them were identified in a new allele for the fumC gene that originated the new ST2528; in addition, 2 isolates were registered as ST2524 and ST2525 through new combination of alleles. Through the MLST database we found that ST656 (CC10) and ST8 (CC165) have a higher homology to ST2524 and ST2525, respectively. However, by the definition of CCs, ST2524 and ST2525 most likely belong to CC10 and CC165, respectively. Our data shows the presence of ESBL producing E. coli isolates in pigs slaughtered for human consumption and raises important questions in the potential risk factors to public health due to the transmission of bacteria carrying resistance through the food chain, and spreading resistance to other bacteria of human clinical significance. A great heterogeneity of MLST types was observed, among which CC10, CC155 and CC101 have already been associated with human clinical isolates

Occurrence of extended-spectrum beta-lactamases in Salmonella enterica strains isolated from broilers and food of animal origin in Portugal

Salmonella enterica is a zoonotic bacteria transmitted through the food chain and isolates harbouring extended-spectrum beta-lactamases (ESBLs) have emerged worldwide during the last decade, with the CTX-M group being particularly important. The aim of the present study was to determine the antimicrobial susceptibility of S. enterica strains isolated from broilers and food of animal origin and to characterize ESBLs producers. On the scope of the national antimicrobial resistance surveillance programme on Salmonella, a total of 283 strains isolated from broilers (n=100) and food of animal origin (n=183), were received at the National Laboratory of Veterinary Research in 2011. The minimum inhibitory concentration (MIC) of 11 antimicrobials (nalidixic acid, ciprofloxacin, ampicillin, cefotaxime, chloramphenicol, florfenicol, streptomycin, gentamicin, tetracycline, sulphamethoxazole and trimethoprim) for all isolates was determined by agar dilution method. Susceptibility towards cefoxitin was determined through disk diffusion method. Breakpoints were interpreted accordingly to EUCAST epidemiological cut-off values. ‘Non-wild type’ (‘NWT’) isolates for cefotaxime (MIC>0.5mg/L) and cefoxitin (<19mm) were screened for the presence of ESBL- (blaTEM, blaOXA, blaSHV, blaCTX) and PMA_-encoding genes, using PCR method. Sequencing was applied to fully identify beta-lactamases. Among broilers, we identified 62% of ‘NWT’ isolates for ciprofloxacin, 57% for nalidixic acid and 28% for sulphamethoxazole, whereas in isolates from food of animal origin, 71%, 63% and 56% were ‘NWT’ isolates for tetracycline, sulphamethoxazole and ampicillin, respectively. Among all, 5/283 (1.8%) strains presented ‘NWT’ MICs for cefotaxime and were multidrug resistant: 2 Salmonella Havana isolated from broilers and 3 Salmonella S. 4,[5],12:i:- isolated from food of animal origin (swine); these isolates had one blaCTX-M-type gene, and 2 from food of animal origin presented 1 blaTEM-type gene and 1 blaSHV-type gene, respectively; they were ‘wild type’ for cefoxitin and no PMAB-encoding gene was detected. To our knowledge, this is the first time in Portugal that ESBL-encoding genes, particularly from blaCTXM family, were detected in isolates of Salmonella Havana, a very common serotype isolated from our broiler population. It should also be emphasised that third generation cephalosporins are not allowed in the national poultry production, contrary to the large animal production, which may explain the detection of ESBL-encoding genes in our strains from swine origin. Horizontal gene transfer may be responsible for the coresistance of strains to non-beta-lactam antibiotics. This study shows that national animal health monitoring systems play an important role and should be improved in an international level

Antimicrobial susceptibility of Salmonella enterica isolated from food-producing animals, animal feed and food products of animal origin, in Portugal - Genetic analysis of isolates with reduced susceptibility/resistance to third generation cephalosporins and cephamycins

Salmonella is a widely distributed foodborne pathogen and one of the most common causes of bacterial foodborne illnesses in humans. An epidemiologic study was conducted on 1600 Salmonella spp isolates recovered from poultry, swine, other animal species, animal feed and food products of animal origin, over the period of 2009-2013, to determine their serotype and antimicrobial susceptibility to a panel of ten antimicrobials (ampicillin, cefotaxime, ciprofloxacin, nalidixic acid, trimethoprim, sulfamethoxazole, chloramphenicol, streptomycine, gentamicine and tetracycline), through the determination of Minimum Inhibitory Concentration (MIC), using the agar dilution technique. Molecular characterization of isolates showing a non-wild type MIC to cefotaxime was performed, to determine extended spectrum β-lactamases (ESBL), plasmid mediated AmpC (PMAβ), plasmid mediated quinolone (PMQR) resistance determinants and mobile genetic elements involved in the dissemination of resistance genes. In live poultry (breeders, broilers, layers) of the 843 isolates recovered, 27.9% comprised S. Enteritidis, 23,5% Salmonella Havana and 14.1% Salmonella Mbandaka; in turkeys, Salmonella Derby was the most common serovar isolated (44%), followed by Salmonella I 4,[5],12:i:- (16%). In swine, of 101 isolates 21.8% comprised Salmonella Rissen and Salmonella Typhimurium, 10.9% Salmonella Derby and Salmonella London. In other animal species, Salmonella Typhimurium was the prevalent serovar with 65.6% of the isolates, followed by Salmonella I 4,[5],12:i:- (9.8%). Overall, S. 4,[5],12:i:- was the most common serotype recovered from food products (25.8%), followed by S. Typhimurium (19.2%) and Salmonella Rissen (18.4%). S. Enteritidis was the most frequent serotype in poultry products (36.3%). Susceptibility profiles differed according with the serotype and the origin of the isolates. A higher frequency of multidrug resistant isolates was recovered from food of swine and bovine origin, with 62.6% and 59.4%, respectively. Polymerase chain reaction and sequencing of the amplicons confirmed the presence of blaCTX-M-type (n=8), blaSHV-type (n=2), blaTEM-type (n=2) and plasmid-mediated AmpC β-lactamases (PMAβ) genes (n=2). No plasmid mediated quinolone resistance-encoding genes were detected. Six isolates( three S. I 4,[5],12:i:-, two S. Havana and one S. Enteritidis) carried class 1 integrons and one S. I 4,[5],12:i:- isolate harboured a class 2 integron. In conclusion, the growing concern of the emergence of bacterial strains bearing ESBL in food-producing animals highlights the importance of continuous monitoring.info:eu-repo/semantics/publishedVersio

Diversity of β-lactamase-encoding genes in Escherichia coli strains isolated from food-producing, companion and zoo animals in Portugal

A rapid development of plasmid-mediated resistance to extended-spectrum cephalosporins has been observed in Enterobacteriaceae worldwide, predominantly due to the dissemination of extended-spectrum beta-lactamases (ESBL) and plasmid-mediated AmpC beta-lactamases (PMAB). The aim of the present study was to evaluate the extension of ESBL- and PMAB-producing E. coli strains isolated from different animal origins in Portugal. For surveillance purposes, 376 E. coli isolates identified at National Laboratory of Veterinary Research (2009-2011) were submitted to antimicrobial susceptibility testing: 123, 51 and 202 were isolated from food-producing, companion and zoo animals, respectively. Minimum Inhibitory Concentrations (MIC) of 11 antimicrobials for all isolates was determined through agar dilution method. Susceptibility towards cefoxitina was determined through disk diffusion method. Breakpoints were interpreted accordingly to EUCAST epidemiological cut-off values. ‘Non-wild type’ (NWT) isolates for cefotaxime (MIC>0.25mg/L) and/or cefoxitina (<19mm) were screened for the presence of ESBL (blaTEM, blaOXA, blaSHV, blaCTX) and PMAB encoding genes, using PCR method. Sequencing was applied to fully identify beta-lactamases. Seventeen isolates (4.5%) were ‘NWT’ strains for cefotaxime, being 5 (29.4%) from companion animals, 4 (23.5%) from food-producing animals and 8 (47.1%) from zoo animals. We identified blaCTX-M-14 (n=1) in a dog and blaCTX-M-15-type genes (n=9) in 6 zoo animals and 3 in food-producing animals. We also identified blaCMY-type genes (n=3) in ‘NWT’ isolates for cefoxitin, one from each animal category. Other beta-lactamase encoding genes were identified: blaOXA in 5 strains (29.4%) isolated from dolphins, blaTEM in 7 strains (41.2%) isolated from 3 companion animals, 2 food-producing and 2 zoo animals, and blaSHV identified in one isolate (5.9%) from a zoo animal; 13 beta-lactamase-producing isolates (76.5%) were multidrug resistant. Among ‘NWT’ E. coli isolates for cefotaxime, we identified an important diversity of ESBL encoding genes, belonging to different families, being blaCTX-M-15-type gene the predominant. The spread of ESBL-producing bacteria among species from different origins, such as food-producing, companion and zoo animals, is a concern at public health level. Thus, it should be a priority to monitor and identify the reservoirs of antimicrobial resistance, contributing to a single health for all