Complete Sequence of a blaOXA-48-Harboring IncL Plasmid from an Enterobacter cloacae Clinical Isolate

Free PMC Article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574381/We report a 63,584-bp conjugative IncL plasmid (pUR17313-1) from an Enterobacter cloacae clinical isolate, containing a blaOXA-48 gene. The plasmid sequence also carried important mobile genetic elements involved in the spread of antibiotic resistance, namely, the Tn1999.2 composite transposon, which enclosed blaOXA-48-, integrase-, and transposase-encoding genes.V.M. was supported by grant number SFRH/BPD/77486/2011 from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal. This study was supported financially by the 2015DDI1228 project from National Institute of Health, Portugal

Occurrence of beta-lactamases, namely GES-5 carbapenemase, among Gram-negative isolates from wastewater samples in Northern Portugal

Antimicrobial resistant pathogens are profoundly relevant to human health and many were the studies that focused on their spread. However, natural and human associated environmental reservoirs of resistance are yet poorly understood. The main goal of this study was to evaluate the main antibiotic resistance mechanisms in Gram-negative bacteria isolates from different wastewater 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 48 Gram-negative isolates (20 Escherichia coli, 8 Citrobacter spp, 7 Klebsiella spp, 6 Kluyvera spp, 2 Enterobacter spp, 1 Hafnia alvei, 1 Pantoea agglomerans, 1Pseudomonas luteola, 1Roultella ornithinolytica, 1Serratia spp) 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 bla and plasmid-mediated quinolone resistance (PMQRs) genes. All isolates were also screened for the presence of class 1 integrons. Overall, 29.2% of the isolates were multidrug resistant, suggesting a great diversity of resistance mechanisms. Noteworthy, 2 isolates showed non-susceptibility to carbapenems, which constitutes one of the last resorts on the antimicrobial therapy. Their phenotypic and molecular characterization revealed the expression of a chromosomal metalo-beta-lactamase in P. luteola and the presence of a GES-5 encoding gene in a Klebsiella pneumoniae isolate. Furthermore, we detected a vast variety of beta-lactamase encoding genes, specifically 12 blaTEM-1 with distinct promoters, 4 blaSHV (2 blaSHV-1 and 2 blaSHV-11), besides different chromossomal AmpC beta-lactamases, namely CMY-65. Class 1 integrons were detected among 6 of TEM-1-producing isolates. Together, these beta-lactamases explain the level of beta-lactam resistance. None PMQR genes were detected. In conclusion, this study provides the first description of a class A carbapenemase in an environmental setting in Portugal, in addition to several other beta-lactam resistance mechanisms. The study highlights the need of surveillance of these resistance mechanisms in environmental backgrounds, since it represents a liable reservoir of potential pathogenic resistant bacteria

Contribution of β-lactamases, porins and efflux pumps to carbapenem and/or fluoroquinolone resistance in clinical isolates

Introduction: Carbapenems, a class of β-lactam antibiotics with very broad activity, are often last resort antibiotics used to treat infections due to extended-spectrum β-lactamase (ESBL)- or plasmid-mediated AmpC (PMAβ)-producing Gram-negative bacteria. Considering the emerging rates of resistance to these antibiotics, the investigation of related resistance mechanisms constitutes an important assignment. Thus, this study aimed to characterize the carbapenem resistance mechanisms harbored by Gram-negative isolates and the co-resistance to structurally unrelated antibiotics (such as fluoroquinolones). Methods: A total of 426 isolates (2 Acinetobacter baumannii, 1 Citrobacter freundii, 7 Enterobacter spp., 153 Escherichia coli, 251 Klebsiella spp., 3 Morganella morganii, 6 Proteus spp., 3 Pseudomonas spp.) were screened for the susceptibility to ertapenem by disc diffusion, and interpreted by SFM guidelines. Non-susceptible isolates were retained and studied against other classes of antibiotics, using the same phenotypic method. Mechanisms justifying the resistance to carbapenemes were searched: 1) carbapenemase production by using molecular methods and isoelectric focusing; 2) modification of outer membrane porins (OMPs) after migration in SDS-PAGE (OmpK35/OmpK36/OmpK37 for 15 Klebsiella pneumoniae, OmpC/OmpF for 1 E. coli, 1 Enterobacter cloacae and 1 Enterobacter cancerogenus, Omp35/Omp36 for 4 Enterobacter aerogenes) and complete characterization of OMP-encoding genes, performed by PCR amplification and sequencing. Deduced amino acid modifications were interpreted by comparing both clinical and wild-type sequences, using the EMBL database. In addition to carbapenemase-encoding genes, other bla, as well as plasmid-mediated quinolone-resistance (PMQR)-encoding genes were also searched by PCR and sequencing. Results: Among the isolates studied, 22 (5%) revealed to be non-susceptible to ertapenem of which 4% were multidrug resistant. Their antibiotic susceptibility evaluation enabled the prediction of the respective resistance mechanisms and guided the remaining biochemical and molecular studies. We identified and characterized the expression of carbapenemases (3 KPC-3 and 1 GES-5), PMAβ (MIR-type) and ESBLs CTX-M-15 (n=11), co-expressed with OXA-1 and TEM-1, and one SHV-12 plus GES-5. Amino acid substitutions were identified in 21 out of 22 isolates, due to insertions, deletions and/or mutations in the nucleotide sequence of OMP-encoding genes in the main regions related to the porin functions, suggesting a role in carbapenem resistance. The acetyltransferase Aac(6’)-Ib-cr variant (n=8) and the efflux pump OqxAB (n=5) were also identified, contributing to fluoroquinolone resistance. Conclusions: Globally, this study provides meaningful insights towards the understanding of emergent carbapenem resistance, as well as PMQR mechanisms, particularly in a worrying multidrug resistant scenario

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)

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

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

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

Predominance of KPC-3 in a Survey for Carbapenemase-Producing Enterobacteriaceae in Portugal

Free PMC Article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432220/Among 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.V. Manageiro was supported by grant SFRH/BPD/77486/2011 from the Fundação para a Ciência e a Tecnologia, Lisbon, Portugal. J. Almeida was supported by grant BRJ-SUB/01/2012 from the National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal. R. A. Bonomo was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under awards R01 AI100560 and R01 AI063517, and by the Cleveland Department of Veterans Affairs, the Veterans Affairs Merit Review Program, and the Geriatric Research Education and Clinical Center VISN 10. We thank the Fundação para a Ciência e a Tecnologia (FCT) for project grant PEst-OE/AGR/UI0211/2011-2014, Strategic Project UI211- 2011-2014

Two Novel CMY-2-Type β-Lactamases Encountered in Clinical Escherichia Coli Isolates

BACKGROUND: Chromosomally encoded AmpC β-lactamases may be acquired by transmissible plasmids which consequently can disseminate into bacteria lacking or poorly expressing a chromosomal bla AmpC gene. Nowadays, these plasmid-mediated AmpC β-lactamases are found in different bacterial species, namely Enterobacteriaceae, which typically do not express these types of β-lactamase such as Klebsiella spp. or Escherichia coli. This study was performed to characterize two E. coli isolates collected in two different Portuguese hospitals, both carrying a novel CMY-2-type β-lactamase-encoding gene. FINDINGS: Both isolates, INSRA1169 and INSRA3413, and their respective transformants, were non-susceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime, but susceptible to cefepime and imipenem, and presented evidence of synergy between cloxacilin and cefoxitin and/or ceftazidime. The genetic characterization of both isolates revealed the presence of bla CMY-46 and bla CMY-50 genes, respectively, and the following three resistance-encoding regions: a Citrobacter freundii chromosome-type structure encompassing a blc-sugE-bla CMY-2-type -ampR platform; a sul1-type class 1 integron with two antibiotic resistance gene cassettes (dfrA1 and aadA1); and a truncated mercury resistance operon. CONCLUSIONS: This study describes two new bla CMY-2-type genes in E. coli isolates, located within a C. freundii-derived fragment, which may suggest their mobilization through mobile genetic elements. The presence of the three different resistance regions in these isolates, with diverse genetic determinants of resistance and mobile elements, may further contribute to the emergence and spread of these genes, both at a chromosomal or/and plasmid level

Characterization of the inhibitor-resistant SHV β-lactamase SHV-107 in a clinical Klebsiella pneumoniae strain co-producing GES-7 enzyme

The clinical Klebsiella pneumoniae INSRA6884 strain exhibited nonsusceptibility to all penicillins tested (MICs of 64 to>2,048 g/ml). The MICs of penicillins were weakly reduced by clavulanate (from 2,048 to 512 g/ml), and tazobactam restored piperacillin susceptibility. Molecular characterization identified the genes blaGES-7 and a new -lactamase gene, blaSHV-107, which encoded an enzyme that differed from SHV-1 by the amino acid substitutions Leu35Gln and Thr235Ala. The SHV-107-producing Escherichia coli strain exhibited only a -lactam resistance phenotype with respect to amoxicillin, ticarcillin, and amoxicillinclavulanate combination. The kinetic parameters of the purified SHV-107 enzyme revealed a high affinity for penicillins. However, catalytic efficiency for these antibiotics was lower for SHV-107 than for SHV-1. No hydrolysis was detected against oxyimino- -lactams. The 50% inhibitory concentration (IC50) for clavulanic acid was 9-fold higher for SHV-107 than for SHV-1, but the inhibitory effects of tazobactam were unchanged. Molecular dynamics simulation suggested that the Thr235Ala substitution affects the accommodation of clavulanate in the binding site and therefore its inhibitory activity.This work was supported financially by the project POCTI/ESP/43037 from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal, awarded to M. Caniça, and by a grant from INRA and Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche (Paris, France), awarded to R. Bonnet. V. Manageiro was supported by grant SFRH/BD/32578/2006 from Fundação para a Ciência e a Tecnologia, Lisbon,Portugal