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

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

The highly conserved serine threonine kinase StkP of Streptococcus pneumoniae contributes to penicillin susceptibility independently from genes encoding penicillin-binding proteins

<p>Abstract</p> <p>Background</p> <p>The serine/threonine kinase StkP of <it>Streptococcus pneumoniae </it>is a major virulence factor in the mouse model of infection. StkP is a modular protein with a N-terminal kinase domain a C-terminal PASTA domain carrying the signature of penicillin-binding protein (PBP) and prokaryotic serine threonine kinase. In laboratory cultures, one target of StkP is the phosphoglucosamine mutase GlmM involved in the first steps of peptidoglycan biosynthesis. In order to further elucidate the importance of StkP in <it>S. pneumoniae</it>, its role in resistance to β-lactams has been assessed by mutational analysis in laboratory cultures and its genetic conservation has been investigated in isolates from infected sites (virulent), asymptomatic carriers, susceptible and non-susceptible to β-lactams.</p> <p>Results</p> <p>Deletion replacement mutation in <it>stkP </it>conferred hypersensitivity to penicillin G and was epistatic on mutations in PBP2X, PBP2B and PBP1A from the resistant 9V clinical isolate URA1258. Genetic analysis of 55 clinical isolates identified 11 StkP alleles differing from the reference R6 allele. None relevant mutation in the kinase or the PASTA domains were found to account for susceptibility of the isolates. Rather the minimal inhibitory concentration (MIC) values of the strains appeared to be determined by their PBP alleles.</p> <p>Conclusion</p> <p>The results of genetic dissection analysis in lab strain Cp1015 reveal that StkP is involved in the bacterial response to penicillin and is epistatic on mutations PBP 2B, 2X and 1A. However analysis of the clinical isolates did not allow us to find the StkP alleles putatively involved in determining the virulence or the resistance level of a given strain, suggesting a strong conservation of StkP in clinical isolates.</p

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

A Comprehensive Review on the Medicinal Plants from the Genus Asphodelus

Free PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874609/Abstract: Plant-based systems continue to play an essential role in healthcare, and their use by different cultures has been extensively documented. Asphodelus L. (Asphodelaceae) is a genus of 18 species and of a total of 27 species, sub-species and varieties, distributed along the Mediterranean basin, and has been traditionally used for treating several diseases particularly associated with inflammatory and infectious skin disorders. The present study aimed to provide a general review of the available literature on ethnomedical, phytochemical, and biological data related to the genus Asphodelus as a potential source of new compounds with biological activity. Considering phytochemical studies, 1,8-dihydroxyanthracene derivatives, flavonoids, phenolic acids and triterpenoids were the main classes of compounds identified in roots, leaf and seeds which were correlated with their biological activities as anti-microbial, anti-fungal, anti-parasitic, cytotoxic, anti-inflammatory or antioxidant agents.The authors wish to thank the Fundação para a Ciência e Tecnologia (FCT) for financial support of iMed.ULisboa project (UID/DTP/04138/2013) as well as a doctoral fellowship granted to the first author (SFRH/BD/125310/2016). This work was supported by the National Research Foundation (FCT), in Portugal, having no role in the data collection and analysis, interpretation of the findings, preparation of the manuscript, or the decision to submit the manuscript for publication.info:eu-repo/semantics/publishedVersio

Diversity of β-lactamase-encoding genes among Gram-negative isolates from water samples in Northern 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 non- pathogenic environmental organisms, where plasmids encoding multiple ARG are prevalent, can provide resistance to most classes of antimicrobials including :-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 plasmid- mediated 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 :-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

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

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)