scholarly journals Contribution of OmpK36 to carbapenem susceptibility in KPC-producing Klebsiella pneumoniae

2009 ◽  
Vol 58 (10) ◽  
pp. 1303-1308 ◽  
Author(s):  
David Landman ◽  
Simona Bratu ◽  
John Quale
Keyword(s):  
New York ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Ionization Mass ◽  
Rt Pcr ◽  
Clonal Group ◽  
Expression Of Genes ◽  
Sds Page ◽  
Genes Encoding ◽  
Zones Of Inhibition

Isolates of Klebsiella pneumoniae harbouring the carbapenemase KPC may have carbapenem MICs that remain in the susceptible range, and may therefore go unrecognized. To understand the mechanisms contributing to the variability in carbapenem MICs, 20 clinical isolates, all belonging to either of two clonal groups of KPC-possessing K. pneumoniae endemic to New York City, were examined. Expression of genes encoding KPC, the porins OmpK35 and OmpK36, and the efflux pump AcrAB was examined by real-time RT-PCR. Outer-membrane profiles of selected KPC-producing isolates were examined by SDS-PAGE, and proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry. The identification of SHV and TEM β-lactamases and the genomic sequences of ompK35 and ompK36 were determined by PCR and DNA sequencing, respectively. For one clonal group, carbapenem MICs increased with decreasing expression of ompK36. A second clonal group also had carbapenem MICs that correlated with ompK36 expression. However, all of the isolates in this latter group continued to produce OmpK36, suggesting that porin configuration may affect entry of carbapenems. For isolates that had the greatest expression of ompK36, carbapenem MICs tended to be lower when determined by the broth microdilution technique, and scattered colonies were seen around the Etest zones of inhibition. All of the KPC-producing isolates were highly resistant to ertapenem, regardless of ompK36 expression. In conclusion, isolates of KPC-possessing K. pneumoniae that express ompK36 tend to have lower MICs to carbapenems and therefore may be more difficult to detect by clinical laboratories. Regardless of ompK36 expression, all of the KPC producers were consistently resistant to ertapenem.

scholarly journals Strain-specific differences in age-related changes in rat susceptibility to experimental autoimmune encephalomyelitis and dendritic cell cytokine gene expression

Genetika ◽  
2014 ◽  
Vol 46 (1) ◽  
pp. 287-301 ◽  
Author(s):  
Biljana Bufan ◽  
Jasmina Djikic ◽  
Mirjana Nacka-Aleksic ◽  
Zorica Stojic-Vukanic ◽  
Mirjana Dimitrijevic ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Rt Pcr ◽  
Autoimmune Encephalomyelitis ◽  
Expression Of Genes ◽  
Age Related ◽  
Genes Encoding ◽  
Immunosuppressive Cytokines ◽  
Age Related Changes ◽  
Experimental Autoimmune

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis, a prototype of Th1/Th17-mediated organ-specific autoimmune disease. In the rat, susceptibility to development of these diseases is shown to be strain-and age-dependent. In adult rats of distinct strains, it correlates with splenic dendritic cell (DC) subset composition, which also exhibit age-related changes. The aim of this study was to examine influence of aging on: i) Albino Oxford (relatively resistant to EAE) and Dark Agouti (susceptible to EAE) rat development of EAE and ii) their splenic conventional (OX62+) DC population in respect to its subset composition and expression of mRNAs for proinflammatory and immunosuppressive cytokines. We used 3month-old (young) and 26-month-old (aged) rats of AO and DA strain. The rats were immunized for EAE with rat spinal cord homogenate in complete Freund?s adjuvant and clinical course of the disease was followed. Fresh OX62+DCs were examined for the expression of CD4 (using flow cytometry) and genes encoding cytokines influencing DC activation/maturation (TNF-? and IL-6) using RT-PCR. Additionally, in vitro lipopolysaccharide (LPS) activated/matured DCs were examined for the expression of genes encoding cytokines controlling Th1/Th17 cell polarization using RT-PCR. With aging, AO rats became more susceptible, whereas DA rats largely lose their susceptibility to the induction of EAE. In AO rats aging shifted CD4+:CD4DC ratio towards CD4-cells, producing large amount of proinflammatory cytokines, whereas in DA rats CD4+:CD4-DC ratio remained stable with aging. In fresh DCs from rats of both the strains the expression of TNF-? mRNA increased with aging, whereas that of IL-6 mRNA decreased and increased in DCs from AO and DA rats, respectively. Following in vitro LPS stimulation OX62+ DCs from aged AO rats up-regulated the expression of mRNA for IL-23p19 (specific subunit of IL-23; crucial for sustained IL-17 production) and IL-1? (positive IL-17 regulator), whereas down-regulated the expression of IL-10 (negative IL-17 regulator) when compared with young strain-matched rats. In DA rats aging incresed IL-23p19 mRNA expression in LPS-stimulated DCs, whereas exerted the opposing effects on the expression of mRNAs for IL-10 and IL-1? compared to AO rats. Irrespective of the rat strain, aging did not influence mRNA expression for IL-12p35 (driving Th1 polarization) in DCs. Overall, results suggest role of changes in the expression of genes encoding proinflammatory and immunosuppressive cytokines in development of age-related alterations in rat susceptibility to EAE induction.

scholarly journals Frequency and expression of mutacin biosynthesis genes in isolates of Streptococcus mutans with different mutacin-producing phenotypes

2008 ◽  
Vol 57 (5) ◽  
pp. 626-635 ◽  
Author(s):  
Regianne Umeko Kamiya ◽  
José Francisco Höfling ◽  
Reginaldo Bruno Gonçalves
Keyword(s):  
Streptococcus Mutans ◽  
Staphylococcus Epidermidis ◽  
High Frequency ◽  
Phenotypic Diversity ◽  
Genetic Determinants ◽  
Rt Pcr ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Spearman Correlation Test ◽  
Expression Frequency

The aim of this study was to analyse the frequency and expression of biosynthesis genes in 47 Streptococcus mutans isolates with different mutacin-producing phenotypes. Detection of the frequency and expression of genes encoding mutacin types I, II, III and IV were carried out by PCR and semi-quantitative RT-PCR, respectively, using primers specific for each type of biosynthesis gene. In addition, a further eight genes encoding putative bacteriocins, designated bsm 283, bsm 299, bsm 423, bsm 1889c, bsm 1892c, bsm 1896, bsm 1906c and bsm 1914, were also screened. There was a high phenotypic diversity; some Streptococcus mutans isolates presented broad antimicrobial spectra against other Streptococcus mutans clinical isolates, including bacteria resistant to common antibiotics, as well as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Streptococcus pyogenes. The expression frequency of the bsm gene was higher than that of the previously characterized mutacins (I–IV). There was no positive correlation between the number of indicator strains inhibited (antimicrobial spectra) and the number of biosynthesis genes expressed (Spearman correlation test, r=−0.03, P>0.05). In conclusion, the high diversity of mutacin-producing phenotypes, associated with high frequency of expression of the biosynthesis genes screened, reveals a broad repertoire of genetic determinants encoding antimicrobial peptides that can act in different combinations.

scholarly journals The Purine Efflux Pump PbuE in Bacillus subtilis Modulates Expression of the PurR and G-Box (XptR) Regulons by Adjusting the Purine Base Pool Size

2005 ◽  
Vol 187 (2) ◽  
pp. 791-794 ◽  
Author(s):  
Per Nygaard ◽  
Hans H. Saxild
Keyword(s):  
Bacillus Subtilis ◽  
De Novo ◽  
Efflux Pump ◽  
Purine Base ◽  
Purine Bases ◽  
Purine Analogs ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Transcriptional Fusions

ABSTRACT In Bacillus subtilis, the expression of genes encoding enzymes and other proteins involved in purine de novo synthesis and salvage is affected by purine bases and phosphoribosylpyrophosphate (PRPP). The transcription of the genes belonging to the PurR regulon is negatively regulated by the PurR protein and PRPP. The expression of the genes belonging to the G-box (XptR) regulon, including the pbuE gene, is negatively regulated by a riboswitch-controlled transcription termination mechanism. The G-box regulon effector molecules are hypoxanthine and guanine. pbuE encodes a purine base efflux pump and is now recognized as belonging to a third purine regulon. The expression of the pbuE gene is positively regulated by a riboswitch that recognizes adenine. Here we show that the expression of pbuE′-lacZ transcriptional fusions are induced by adenine to the highest extent in mutants which do not express a functional PbuE pump. In a mutant defective in the metabolism of adenine, the ade apt mutant, we found a high intracellular level of adenine and constitutive high levels of PbuE. A growth test using a purine auxotroph provided further evidence for the role of PbuE in lowering the intracellular concentration of purine bases, including adenine. Purine analogs also affect the expression of pbuE, which might be of importance for the protection against toxic analogs. In a mutant that overexpresses PbuE, the expression of genes belonging to the PurR regulon was increased. Our findings provide further evidence for important functions of the PbuE protein, such as acting as a pump that lowers the purine base pool and affects the expression of the G-box and PurR regulons, including pbuE itself, and as a pump involved in protection against toxic purine base analogs.

scholarly journals The Envelope Proteome Changes Driven by RamA Overproduction in Klebsiella pneumoniae that Enhance Acquired β-Lactam Resistance

2017 ◽  
Author(s):  
Juan-Carlos Jiménez-Castellanos ◽  
Wan Ahmad Kamil Wan Nur Ismah ◽  
Yuiko Takebayashi ◽  
Jacqueline Findlay ◽  
Thamarai Schneiders ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Envelope Protein ◽  
Protein Production ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Protein Abundance ◽  
Rt Pcr ◽  
Proteomics Analysis ◽  
Over Expression ◽  
Proteome Changes

AbstractOBJECTIVESIn Klebsiella pneumoniae, overproduction of RamA results in reduced envelope permeability and reduced antimicrobial susceptibility but clinically relevant resistance is rarely observed. Here we have tested whether RamA over-production can enhance acquired β-lactam resistance mechanisms in K. pneumoniae and have defined the envelope protein abundance changes seen upon RamA overproduction during growth in low and high osmolarity media.METHODSEnvelope permeability was estimated using a fluorescent dye accumulation assay. Antibiotic susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels quantified by Real Time RT-PCR.RESULTSRamA overproduction enhanced β-lactamase mediated β-lactam resistance, in some cases dramatically, without altering β-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF over-expression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 out of 4 carbapenemase producers, 1/21 CTX-M producers and 2/19 strains not carrying CTX-M or carbapenemases.CONCLUSIONSWhilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production.

scholarly journals Activity of Aztreonam in Combination with Avibactam, Clavulanate, Relebactam, and Vaborbactam against Multidrug-Resistant Stenotrophomonas maltophilia

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
M. Biagi ◽  
D. Lamm ◽  
K. Meyer ◽  
A. Vialichka ◽  
M. Jurkovic ◽  
...  
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Molecular Mechanisms ◽  
Efflux Pump ◽  
Treatment Strategies ◽  
Multidrug Resistant ◽  
Content Type ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Novel Treatment Strategies

ABSTRACT The intrinsic L1 metallo- and L2 serine-β-lactamases in Stenotrophomonas maltophilia make it naturally multidrug resistant and difficult to treat. There is a need to identify novel treatment strategies for this pathogen, especially against isolates resistant to first-line agents. Aztreonam in combination with avibactam has demonstrated potential, although data on other aztreonam–β-lactamase inhibitor (BLI) combinations are lacking. Additionally, molecular mechanisms for reduced susceptibility to these combinations have not been explored. The objectives of this study were to evaluate and compare the in vitro activities and to understand the mechanisms of resistance to aztreonam in combination with avibactam, clavulanate, relebactam, and vaborbactam against S. maltophilia. A panel of 47 clinical S. maltophilia strains nonsusceptible to levofloxacin and/or trimethoprim-sulfamethoxazole were tested against each aztreonam-BLI combination via broth microdilution, and 6 isolates were then evaluated in time-kill analyses. Three isolates with various aztreonam-BLI MICs were subjected to whole-genome sequencing and quantitative reverse transcriptase PCR. Avibactam restored aztreonam susceptibility in 98% of aztreonam-resistant isolates, compared to 61, 71, and 15% with clavulanate, relebactam, and vaborbactam, respectively. The addition of avibactam to aztreonam resulted in a ≥2-log10-CFU/ml decrease at 24 h versus aztreonam alone against 5/6 isolates compared to 1/6 with clavulanate, 4/6 with relebactam, and 2/6 with vaborbactam. Molecular analyses revealed that decreased susceptibility to aztreonam-avibactam was associated with increased expression of genes encoding L1 and L2, as well as the efflux pump (smeABC). Aztreonam-avibactam is the most promising BLI-combination against multidrug-resistant S. maltophilia. Decreased susceptibility may be due to the combination of overexpressed β-lactamases and efflux pumps. Further studies evaluating this combination against S. maltophilia are warranted.

scholarly journals Metabolism of the Cyanogenic Glucosides in Developing Flax: Metabolic Analysis, and Expression Pattern of Genes

Metabolites ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 288
Author(s):  
Magdalena Zuk ◽  
Katarzyna Pelc ◽  
Jakub Szperlik ◽  
Agnieszka Sawula ◽  
Jan Szopa
Keyword(s):  
Amino Acids ◽  
Strongly Correlated ◽  
Sulfur Amino Acids ◽  
Rt Pcr ◽  
Cyanogenic Glucosides ◽  
Cyanide Detoxification ◽  
Expression Of Genes ◽  
General Metabolism ◽  
Genes Encoding ◽  
Detoxification Process

Cyanogenic glucosides (CG), the monoglycosides linamarin and lotaustralin, as well as the diglucosides linustatin and neolinustatin, have been identified in flax. The roles of CG and hydrogen cyanide (HCN), specifically the product of their breakdown, differ and are understood only to a certain extent. HCN is toxic to aerobic organisms as a respiratory inhibitor and to enzymes containing heavy metals. On the other hand, CG and HCN are important factors in the plant defense system against herbivores, insects and pathogens. In this study, fluctuations in CG levels during flax growth and development (using UPLC) and the expression of genes encoding key enzymes for their metabolism (valine N-monooxygenase, linamarase, cyanoalanine nitrilase and cyanoalanine synthase) using RT-PCR were analyzed. Linola cultivar and transgenic plants characterized by increased levels of sulfur amino acids were analyzed. This enabled the demonstration of a significant relationship between the cyanide detoxification process and general metabolism. Cyanogenic glucosides are used as nitrogen-containing precursors for the synthesis of amino acids, proteins and amines. Therefore, they not only perform protective functions against herbivores but are general plant growth regulators, especially since changes in their level have been shown to be strongly correlated with significant stages of plant development.

scholarly journals Klebsiella pneumoniae Isolate from a New York City Hospital Belonging to Sequence Type 258 and CarryingblaKPC-2andblaVIM-4

2016 ◽  
Vol 60 (3) ◽  
pp. 1924-1927 ◽  
Author(s):  
Mariana Castanheira ◽  
Lalitagauri M. Deshpande ◽  
Janet C. Mills ◽  
Ronald N. Jones ◽  
Rosemary Soave ◽  
...  
Keyword(s):  
New York ◽  
New York City ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Sequence Type ◽  
City Hospital ◽  
Class 1 Integron ◽  
Content Type ◽  
Class 1 ◽  
Elevated Expression

Among 69 of 139 (49.6%) carbapenem-nonsusceptibleEnterobacteriaceaecarryingblaKPC, 1Klebsiella pneumoniaewas also positive forblaVIM. The isolate belonged to sequence type 258 (ST258) and carriedblaKPC-2on a copy ofTn4401a andblaVIM-4on a class 1 integron. Genes were located on distinct plasmids belonging to Inc types A/C and FII. Elevated expression of the efflux pump AcrAB-TolC (acrA, 15.3 times) and reduced expression of outer membrane protein genesompK35andompK37(0.16 and 0.081 times, respectively) associated with various amino acid alterations on OmpK37 were observed. The presence of two carbapenemases in ST258K. pneumoniaeis of great concern due to the ability of this organism to widely disseminate.

scholarly journals Genetic Organization of Transposase Regions Surrounding blaKPC Carbapenemase Genes on Plasmids from Klebsiella Strains Isolated in a New York City Hospital

2009 ◽  
Vol 53 (5) ◽  
pp. 1998-2004 ◽  
Author(s):  
Thomas D. Gootz ◽  
Mary Kay Lescoe ◽  
Fadia Dib-Hajj ◽  
Brian A. Dougherty ◽  
Wen He ◽  
...  
Keyword(s):  
New York ◽  
New York City ◽  
Klebsiella Pneumoniae ◽  
York City ◽  
Klebsiella Oxytoca ◽  
Promoter Sequence ◽  
The United States ◽  
City Hospital ◽  
Genes Encoding ◽  
Healthcare Associated

ABSTRACT Carbapenem-resistant Klebsiella strains carrying Klebsiella pneumoniae carbapenemases (KPC) are endemic to New York City and are spreading across the United States and internationally. Recent studies have indicated that the KPC structural gene is located on a 10-kb plasmid-borne element designated Tn4401. Fourteen Klebsiella pneumoniae strains and one Klebsiella oxytoca strain isolated at a New York City hospital in 2005 carrying either bla KPC-2 or bla KPC-3 were examined for isoforms of Tn4401. Ten of the Klebsiella strains contained a 100-bp deletion in Tn4401, corresponding to the Tn4401a isoform. The presence of this deletion adjacent to the upstream promoter region of bla KPC in Tn4401a resulted in a different −35 promoter sequence of TGGAGA than that of CTGATT present in isoform Tn4401b. Complete sequencing of one plasmid carrying bla KPC from each of three nonclonal isolates indicated the presence of genes encoding other types of antibiotic resistance determinants. The 70.6-kb plasmid from K. pneumoniae strain S9 carrying bla KPC-2 revealed two identical copies of Tn4401b inserted in an inverse fashion, but in this case, one of the elements disrupted a group II self-splicing intron. In K. pneumoniae strain S15, the Tn4401a element carrying bla KPC-2 was found on both a large 120-kb plasmid and a smaller 24-kb plasmid. Pulsed-field gel electrophoresis results indicate that the isolates studied represent a heterogeneous group composed of unrelated as well as closely related Klebsiella strains. Our results suggest that endemic KPC-positive Klebsiella strains constitute a generally nonclonal population comprised of various alleles of bla KPC on several distinct plasmid genetic backgrounds. This study increases our understanding of the genetic composition of the evolving and expanding role of KPC-producing, healthcare-associated, gram-negative pathogens.

scholarly journals Purification, Identification And Characterization of Nag2 N-Acetylglucosaminidase From Trichoderma Virens Strain Mango

2021 ◽  
Author(s):  
Jheng-Hua Huang ◽  
Feng-Jin Zeng ◽  
Jhe-Fu Guo ◽  
Jian-Yuan Huang ◽  
Hua-Chian Lin ◽  
...  
Keyword(s):  
Sclerotium Rolfsii ◽  
Trichoderma Virens ◽  
Rt Pcr ◽  
Mycelium Growth ◽  
Trichoderma Spp ◽  
Sds Page ◽  
Genes Encoding ◽  
Industrial Use ◽  
Identification And Characterization

Abstract Background: N -acetylglucosaminidase (NAGase) could liberate N -acetylglucosamine (GlcNAc) from GlcNAc-containing oligosaccharides. Trichoderma spp. is an important source of chitinase, particularly NAGase for industrial use. nag1 and nag2 genes encoding NAGase , are found in the genome in Trichoderma spp. The deduced Nag1 and Nag2 shares ~55% homology in Trichoderma virens. Most studies were focus on Nag1 and nag1 previously. Results: The native NAGase (TvmNAG2) was purified to homogeneity with molecular mass of ~68 kDa on SDS-PAGE analysis, and identified as Nag2 by MALDI/MS analysis from an isolate T. virens strain mango. RT-PCR analyses revealed that only nag2 gene was expressed in liquid culture of T. virens , while both of nag1 and nag2 were expressed in T. virens cultured on the plates. TvmNAG2 was thermally stable up to 60 o C for 2 h, and the optimal pH and temperature were 5.0 and 60-65 o C, respectively, using p -nitrophenyl- N -acetyl- β -D-glucosaminide ( p NP-NAG) as substrate. Using colloidal chitin as substrate, the end product catalyzed by TvmNAG2 was GlcNAc, based on HPLC and TLC analyses. The optimal temperature for TvmNAG2 to produce GlcNAc was 40 o C. TvmNAG2 possesses antifungal activity, inhibiting the mycelium growth of Sclerotium rolfsii . And it was resistant to the proteolysis by papain and trypsin. Conclusions: The native Nag2, TvmNAG2 was purified and identified from T. virens strain mango, as well as enzymatic properties. To our knowledge, it is the first report with the properties of native Trichoderma Nag2.

scholarly journals The effect of indole-3-carbinol on the expression of CYP1A1, CYP1B1 and AhR genes and proliferation of MCF-7 cells.

2007 ◽  
Vol 54 (1) ◽  
pp. 113-117 ◽  
Author(s):  
Marta Ociepa-Zawal ◽  
Błazej Rubiś ◽  
Mariusz Laciński ◽  
Wiesław H Trzeciak
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Breast Cancer Cell ◽  
Pcr Analysis ◽  
Estrogen Metabolism ◽  
Rt Pcr ◽  
P21 Protein ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Mcf 7

The influence of an antiestrogen, indole-3-carbinol (I3C) on the expression of CYP1A1, CYP1B1 and AhR genes was investigated in an attempt to establish whether I3C could increase the expression of genes involved in estrone metabolism. Another purpose was to examine the proliferation of an estrogen-dependent breast cancer cell (MCF-7 line) under the influence of I3C and both I3C and DDT. In MCF-7 cells incubated with I3C or I3C and DDT combined, quantitative RT-PCR analysis revealed a significant increase in the level of CYP1A1, AhR, and CYP1B1 transcripts. The proliferation rate of MCF-7 cells was increased by treatment with DDT or estradiol (E2), whereas I3C did not affect the proliferation of MCF-7 cells but greatly reduced the stimulatory effect of DDT, and abolished the effect of E2. The level of p21 transcript, encoding p21 protein involved in the cell cycle, was increased several-fold by I3C comparing to its level in cells incubated with estradiol or DDT. The results suggest that the proliferation of MCF-7 cells is accompanied not only by expression of genes encoding cytochromes involved in estrogen metabolism, but also by changes in the expression of other genes including that encoding p21 protein involved in the cell cycle.

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