scholarly journals First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling.

2020 ◽  
Author(s):  
Xuebin Tian ◽  
Qiongdan Wang ◽  
Laura Perlaza-Jiménez ◽  
Xiangkuo Zheng ◽  
Yajie Zhao ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Gel Electrophoresis ◽  
Stop Codon ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Premature Stop Codon ◽  
Sequencing Analysis ◽  
Membrane Remodeling ◽  
Carbapenem Resistant

Abstract Background: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a looming threat to human health. Although there are numerous studies regarding porin alteration in association with the production of ESBLs and/or AmpC β-lactamase, a systematic study on the treatment-emergence of porins alteration in antibiotic resistance does not yet exist. The aim of this study was to investigate the underlying mechanism of resistance of K. pneumoniae during carbapenem treatment. Results: Here, we report three strains (FK-2624, FK-2723 and FK-2820) isolated from one patient before and after imipenem treatment during hospitalization. Antibiotic susceptibility testing indicated that that the first isolate, FK-2624, was susceptible to almost all tested antimicrobials, being resistant only to fosfomycin. The subsequent isolates FK-2723 and FK-2820 were multidrug resistant (MDR). After imipenem therapy, FK-2820 was found to be carbapenem-resistant. PCR and Genome Sequencing analysis indicated that oqxA, and fosA5, were identified in all three strains. In addition, FK-2624 also harbored blaSHV-187 and blaTEM-116. The blaSHV-187 and blaTEM-116 genes were not detected in FK-2723 and FK-2820. blaDHA-1, qnrB4, aac (6’)-IIc, and blaSHV-12, EreA2, CatA2, SulI, and tetD, were identified in both FK-2723 and FK-2820. Moreover, the genes blaDHA-1, qnrB4, aac (6’)-IIc were co-harbored on a plasmid. Of the virulence factors found in this study, ybtA, ICEKp6, mrkD, entB, iroN, rmpA2-6, wzi16 and capsular serotype K57 were found in the three isolates. The results of pairwise comparisons, multi-locus sequencing typing (MLST) and pulsed-field gel electrophoresis (PFGE) revealed high homology among the isolates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that isolate FK-2820 lacked OmpK36, with genome sequence data validating that there was a premature stop codon in the ompK36 gene and real-time RT-PCR suggesting high turnover of the ompK36 non-sense transcript in FK-2820, with the steady-state mRNA level 0.007 relative to the initial isolate. Conclusion: This study in China highlight that the alteration of outer membrane porins due to the 14-day use of imipenem play a potential role in leading to clinical presentation of carbapenem-resistance. This is the first description of increased resistance developing from a carbapenem-susceptible K. pneumoniae with imipenem treatment driven by outer membrane remodeling.

scholarly journals First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling

2020 ◽  
Author(s):  
Xuebin Tian ◽  
Qiongdan Wang ◽  
Laura Perlaza-Jiménez New ◽  
Xiangkuo Zheng ◽  
Yajie Zhao ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Gel Electrophoresis ◽  
Stop Codon ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Premature Stop Codon ◽  
Sequencing Analysis ◽  
Membrane Remodeling ◽  
Carbapenem Resistant

Abstract Background:The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a looming threat to human health. Although there are numerous studies regarding porin alteration in association with the production of ESBLs and/or AmpC β-lactamase, a systematic study on the treatment-emergence of porins alteration in antibiotic resistance does not yet exist. The aim of this study was to investigate the underlying mechanism and evolution of resistance of K. pneumoniae during carbapenem treatment. Results:Here, we report three strains (FK-2624, FK-2723 and FK-2820) isolated from one patient before and after imipenem treatment during hospitalization. Antibiotic susceptibility testing indicated that that the first isolate, FK-2624, was susceptible to almost all tested antimicrobials, being resistant only to fosfomycin. The subsequent isolates FK-2723 and FK-2820 were multidrug resistant (MDR). After imipenem therapy, FK-2820 had evolved to be carbapenem-resistant. PCR and Genome Sequencing analysis indicated that oqxA, and fosA5, were identified in all three strains. In addition, FK-2624 also harbored bla SHV-187 and blaTEM-116. The blaSHV-187 and blaTEM-116 genes were not detected in FK-2723 and FK-2820. blaDHA-1, qnrB4, aac (6’)-IIc, and blaSHV-12, EreA2, CatA2, SulI, and tetD , were identified in both FK-2723 and FK-2820. Moreover, the genes bla DHA -1, qnrB4, aac(6’)-IIc were co-harbored on a plasmid. Of the virulence factors found in this study, ybtA, ICEKp6, mrkD, entB, iroN, rmpA2-6, wzi16 and capsular serotype K57 were found in the three isolates. The results of pairwise comparisons, multi-locus sequencing typing (MLST) and pulsed-field gel electrophoresis (PFGE) revealed high homology among the isolates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that isolate FK-2820 lacked OmpK36, with genome sequence data validating that there was a premature stop codon in the ompK36 gene and real-time RT-PCR suggesting high turnover of the ompK36 non-sense transcript in FK-2820, with the steady-state mRNA level 0.007 relative to the initial isolate. Conclusion:This study in China highlight that the alteration of outer membrane porins due to the 14-day use of imipenem play a potential role in leading to clinical presentation of carbapenem-resistance. This is the first description of increased resistance developing from a carbapenem-susceptible K. pneumoniae with imipenem treatment driven by outer membrane remodeling.

scholarly journals First description of increased resistance in carbapenem-susceptible Klebsiella pneumoniae with imipenem treatment driven by outer membrane remodelling in China

2019 ◽  
Author(s):  
Xuebin Tian ◽  
Qiongdan Wang ◽  
Xiangkuo Zheng ◽  
Yajie Zhao ◽  
Renchi Fang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Gel Electrophoresis ◽  
Stop Codon ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Premature Stop Codon ◽  
Underlying Mechanism ◽  
Systematic Research ◽  
Carbapenem Resistant ◽  
Before And After

Abstract The emergence of carbapenem-resistant Kelbsiella pneumoniae (CRKP) posed threats to human health. Although there are numerous studies regarding porin alteration in association with the production of ESBLs and/or AmpC β-lactamase, a systematic research about the treatment-emergence of porins alteration in antibiotic resistance does not exist yet. The aim of this study was to investigate the underlying mechanism and evolution of resistance of K. pneumoniae during carbapenem treatment. Here, we reported three strains (FK-2624, FK-2723 and FK-2820) isolated from one patient before and after imipenem treatment during hospitalization. Antibiotic susceptibility testing indicated that FK-2624 was susceptible to almost antimicrobials but fosfomycin; FK-2723 and FK-2820 were MDR. After imipenem therapy, FK-2820 was evolved to carbapenem-resistant. PCR and Whole-Genome sequencing ( WGS) indicated that resistance genes bla SHV , oqxA and fosA5 were detected in FK-2624, in addition, FK-2723 and FK-2820 harbored bla DHA , qnrB , aac (6’)-Ib . Virulence factors K57, ybtA, mrkD, entB and iroN were detected simultaneously in all of three strains. The results of pairwise comparisons , multi-locus sequencing typing (MLST) and pulsed-field gel electrophoresis (PFGE) revealed high homology among the isolates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that isolate FK-2820 lacked OmpK 36 as there was a premature stop codon of the outer membrane porin encoding gene ompk36 confirmed by sequencing. Real-time RT-PCR revealed that the expression of ompK36 in FK-2820 was 0.093 times the control isolate ATCC 13883. Our study highlighted that the alteration of outer membrane porins due to the 14-day use of imipenem clinically play a potential role in leading to the carbapenems-resistance of FK-2820.

Molecular weight determination and partial characterization of Klebsiella pneumoniae hemolysins

1986 ◽  
Vol 32 (11) ◽  
pp. 884-888 ◽  
Author(s):  
Lucila Isabel Barberis ◽  
Alberto Jorge Eraso ◽  
Maria Cristina Pàjaro ◽  
Inès Albesa
Keyword(s):  
Klebsiella Pneumoniae ◽  
Gel Electrophoresis ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Growth Media ◽  
Molecular Weight Determination ◽  
Periodic Acid Schiff

Two thiol-activated Klebsiella pneumoniae hemolysins were purified from growth media by means of salt precipitation, gel filtration, ion-exchange chromatography, and polyacrylamide gel electrophoresis. The hemolysins peaks coincided with the protein and glycoprotein peaks as determined by chromatography and electrophoresis, The molecular weights, estimated by gel filtration, were 8400 and 19 000; by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, the values were calculated as 15 500 and 27 000. The electrophoretic bands were best detected by the periodic acid–Schiff method. Reduction of the disulfide linkages did not cause the originally larger molecule to break into 8400 and 19 000 hemolysins. However, trypsin treatment cleaved the 19 000 hemolysin into an active moiety, with an electrophoretic migration similar to the 8400 hemolysin. A naturally occurring proteolytic activity was investigated using pepstatin and antipain. When the trypsin inhibitor was added to the system, the hemolytic activity was detected only in the 19 000 hemolysin and the smaller hemolysin was absent.

Outer membrane proteins from Serratia marcescens

1993 ◽  
Vol 39 (1) ◽  
pp. 108-111 ◽  
Author(s):  
Marta Puig ◽  
Carme Fusté ◽  
Miquel Viñas
Keyword(s):  
Membrane Proteins ◽  
Serratia Marcescens ◽  
Outer Membrane ◽  
Nutrient Availability ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cultural Conditions

The outer membrane proteins (OMPs) of several strains of Serratia marcescens have been studied by sodium dodecyl sulphate – urea – polyacrylamide gel electrophoresis. Four major OMPs, named Omp1, Omp2, Omp3, and OmpA (42, 40, 39, and 37 kDa, respectively), have been visualized. The relative proportions of Omp2 and Omp3 depend on cultural conditions (temperature of incubation, osmolarity, and nutrient availability).Key words: Serratia marcescens, outer membrane proteins, porin.

Mechanisms of ertapenem resistance in Enterobacteriaceae isolates in a tertiary university hospital

2016 ◽  
Vol 64 (5) ◽  
pp. 1042-1049 ◽  
Author(s):  
Hae-Sun Chung ◽  
Dongeun Yong ◽  
Miae Lee
Keyword(s):  
Gel Electrophoresis ◽  
Molecular Mechanisms ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
University Hospital ◽  
Clinical Microbiology Laboratory ◽  
Sequencing Analysis ◽  
Sds Page ◽  
Broth Microdilution Method

The aim of this study was to investigate the molecular mechanisms of ertapenem resistance among Enterobacteriaceae isolates in a clinical microbiology laboratory at a tertiary university hospital. A total of 40 clinical isolates including 20 resistant and 20 intermediate isolates were collected from August 2012 to July 2013. Ertapenem susceptibility was confirmed by the broth microdilution method. PCR and sequencing analysis of carbapenemase, AmpC β-lactamase, and extended-spectrum β-lactamase (ESBL) genes were performed. Outer membrane proteins (OMPs) were examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Molecular epidemiology studies were performed by pulsed-field gel electrophoresis (PFGE). AmpC β-lactamases and ESBLs were found in 32 (80.0%) and 20 (50.0%) of the 40 isolates with ertapenem non-susceptibility, respectively. Distributions of β-lactamase genes differed among the species. OneCitrobacter freundiiisolate among the 40 isolates with ertapenem non-susceptibility carrying theblaIMP-1associated class 1 integron was detected. SDS-PAGE of OMPs showed altered or greatly diminished expression of porins in all isolates ofKlebsiella pneumoniae(n=5) andEnterobacter cloacae(n=11) with ertapenem resistance. Porin alterations were less common among the isolates with intermediate susceptibility (4/19). Integration of the results of molecular analysis of β-lactamases and OMP analysis revealed that most of the isolates with ertapenem resistance exhibited β-lactamase activity and porin alteration. PFGE revealed that most isolates were epidemiologically unrelated. Ertapenem resistance in clinical Enterobacteriaceae isolates was associated with β-lactamase activity and porin alteration. Even though carbapenemase-producing Enterobacteriaceae are still rare, continuous monitoring and infection control for carbapenem-resistant Enterobacteriaceae are necessary.

scholarly journals Characterization of Extended-Host-Range Pseudo-T-Even Bacteriophage Kpp95 Isolated on Klebsiella pneumoniae

2007 ◽  
Vol 73 (8) ◽  
pp. 2532-2540 ◽  
Author(s):  
Lii-Tzu Wu ◽  
Shu-Ying Chang ◽  
Ming-Ren Yen ◽  
Tsuey-Ching Yang ◽  
Yi-Hsiung Tseng
Keyword(s):  
Klebsiella Pneumoniae ◽  
Host Range ◽  
Gel Electrophoresis ◽  
Phylogenetic Analyses ◽  
Klebsiella Oxytoca ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Amino Acid Residues ◽  
Enterobacter Agglomerans

ABSTRACT Kpp95, isolated on Klebsiella pneumoniae, is a bacteriophage with the morphology of T4-type phages and is capable of rapid lysis of host cells. Its double-stranded genomic DNA (ca. 175 kb, estimated by pulsed-field gel electrophoresis) can be cut only by restriction endonucleases with a cleavage site flanked either by A and T or by T, as tested, suggesting that it contains the modified derivative(s) of G and/or C. Over 26 protein bands were visualized upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the virion proteins. N-terminal sequencing indicated that the most abundant band (46 kDa) is the major coat protein (gp23) which has been cleaved from a signal peptide likely with a length similar to that of T4. Phylogenetic analyses based on the sequences of the central region (263 amino acid residues) of gp23 and the full length of gp18 and gp19 placed Kpp95 among the pseudo-T-even subgroup, most closely related to the coliphage JS98. In addition to being able to lyse many extended-spectrum β-lactamase strains of K. pneumoniae, Kpp95 can lyse Klebsiella oxytoca, Enterobacter agglomerans, and Serratia marcescens cells. Thus, Kpp95 deserves further studies for development as a component of a therapeutic cocktail, owing to its high efficiencies of host lysis plus extended host range.

scholarly journals Rh(D) antigen expression and isolation of a new Rh(D) cDNA isoform in human erythroleukemic K562 cells

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1975-1981 ◽  
Author(s):  
K Suyama ◽  
R Lunn ◽  
S Haller ◽  
J Goldstein
Keyword(s):  
Stop Codon ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Premature Stop Codon ◽  
K562 Cells ◽  
Antigen Expression ◽  
Reading Frame ◽  
Rabbit Reticulocyte Lysate System ◽  
D Antigen

Abstract Human erythroleukemic K562 cells are known to have several erythroid properties. K562 cells possess Rh mRNAs, but expression of Rh proteins has not previously been reported. We immunoprecipitated Rh protein from K562 cell lysate using rabbit anti-Rh and detected Rh(D) antigens on K562 cells using fluorescence-activated cell sorting (FACS). These results suggest that K562 cells will be useful as an expression model for most Rh antigens. We also cloned a new Rh(D) cDNA isoform (RhK562- II), from a K562 cDNA library using polymerase chain reaction (PCR) with 5′ and 3′ end oligonucleotides of the published Rh(e/E) antigen encoding cDNA sequence as primers. Sequence analysis showed that RhK562- II is composed of 951 nucleotides (316 amino acids), identical to the first 939 nucleotides (exons 1 to 6) of one of the Rh(D) cDNAs (RhXIII), except for nucleotide 654 (C-->XG exchange). However, this exchange is the same as that of another published Rh(D) cDNA (RhPII cDNA). RhK562-II is deprived of exons 7 and 8 (nucleotides 940 to 1,153), followed by an identical sequence up to the 3′ end of the open- reading frame of the RhXIII cDNA, which causes a frame-shift mutation and produces a premature stop codon. In vitro expression of RhK562-II using the transcription and translation rabbit reticulocyte lysate system produced two major Rh-related proteins (30 kD and 25 kD), which were immunoprecipitated by rabbit polyclonal anti-Rh and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

scholarly journals Rh(D) antigen expression and isolation of a new Rh(D) cDNA isoform in human erythroleukemic K562 cells

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1975-1981 ◽  
Author(s):  
K Suyama ◽  
R Lunn ◽  
S Haller ◽  
J Goldstein
Keyword(s):  
Stop Codon ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Premature Stop Codon ◽  
K562 Cells ◽  
Antigen Expression ◽  
Reading Frame ◽  
Rabbit Reticulocyte Lysate System ◽  
D Antigen

Human erythroleukemic K562 cells are known to have several erythroid properties. K562 cells possess Rh mRNAs, but expression of Rh proteins has not previously been reported. We immunoprecipitated Rh protein from K562 cell lysate using rabbit anti-Rh and detected Rh(D) antigens on K562 cells using fluorescence-activated cell sorting (FACS). These results suggest that K562 cells will be useful as an expression model for most Rh antigens. We also cloned a new Rh(D) cDNA isoform (RhK562- II), from a K562 cDNA library using polymerase chain reaction (PCR) with 5′ and 3′ end oligonucleotides of the published Rh(e/E) antigen encoding cDNA sequence as primers. Sequence analysis showed that RhK562- II is composed of 951 nucleotides (316 amino acids), identical to the first 939 nucleotides (exons 1 to 6) of one of the Rh(D) cDNAs (RhXIII), except for nucleotide 654 (C-->XG exchange). However, this exchange is the same as that of another published Rh(D) cDNA (RhPII cDNA). RhK562-II is deprived of exons 7 and 8 (nucleotides 940 to 1,153), followed by an identical sequence up to the 3′ end of the open- reading frame of the RhXIII cDNA, which causes a frame-shift mutation and produces a premature stop codon. In vitro expression of RhK562-II using the transcription and translation rabbit reticulocyte lysate system produced two major Rh-related proteins (30 kD and 25 kD), which were immunoprecipitated by rabbit polyclonal anti-Rh and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

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