Özden Baltekin | Uppsala University (original) (raw)

Papers by Özden Baltekin

Blood stream infection (BSI) is related to high mortality and morbidity. Early antimicrobial ther... more Blood stream infection (BSI) is related to high mortality and morbidity. Early antimicrobial therapy is crucial in treating patients with BSI. The most common Gram-negative bacteria causing BSI is Escherichia coli. Targeted effective treatment of patients with BSI is only possible if it is based on antibiotic susceptibility testing (AST) data after blood culture positivity. However, there are very few methods available for rapid phenotypic AST and the fastest method takes 4 h. Here we analyzed the performance of a 30 min ultra-rapid method for AST of E. coli directly from positive blood cultures (BC). In total, 51 positive BC with E. coli were studied, and we evaluated the ultra-rapid method directly on positive BC as well as on E. coli colonies cultured on agar plates. The results obtained by the new method were compared with disk diffusion. The method provided accurate AST result in 30 min to Ciprofloxacin and Gentamicin for 92% and 84% of the positive BC samples, respectively. Fo...

arXiv: Quantitative Methods, 2015

Isogenic Escherichia coli growing exponentially in a constant environment display large variation... more Isogenic Escherichia coli growing exponentially in a constant environment display large variation in growth-rates, division-sizes and generation-times. It is unclear how these seemingly random cell cycles can be reconciled with the precise regulation required under conditions where the generation time is shorter than the time to replicate the genome. Here we use single molecule microscopy to map the location of the replication machinery to the division cycle of individual cells. We find that the cell-to-cell variation in growth rate is sufficient to explain the corresponding variation in cell size and division timing assuming a simple mechanistic model. In the model, initiation of chromosome replication is triggered at a fixed volume per origin region, and associated with each initiation event is a division event at a growth rate dependent time later. The result implies that cell division in E. coli has no other regulation beyond what is needed to initiate DNA replication at the rig...

Gene regulation mediated by transcription factors (TFs) is essential for all organisms. The funct... more Gene regulation mediated by transcription factors (TFs) is essential for all organisms. The functionality of TFs can largely be described by the fraction of time they occupy their regulatory binding sites on the chromosome. DNA-binding proteins have been shown to find their targets through facilitated diffusion in vitro. In its simplest form this means that the protein combines a random 3D search in the cytoplasm with 1D sliding along DNA. This has been proposed to speed up target location. It is difficult to mimic the in vivo conditions for gene regulation in biochemistry experiments; i.e. the ionic strength, chromosomal structure, and the presence of other DNA-binding macromolecules. In this thesis single molecule imaging assays for live cell measurements were developed to study the kinetics of the Escherichia coli transcription factor LacI. The low copy number LacI, in fusion with a fluorescent protein (Venus) is detected as a localized near-diffraction limited spot when being DN...

The EMBO journal, Jan 2, 2018

Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. ... more Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing...

So far, it has not been possible to perform advanced microscopy on pool generated strain librarie... more So far, it has not been possible to perform advanced microscopy on pool generated strain libraries and at the same time know each strain’s genotype. We have overcome this barrier by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single molecule fluorescence imaging of E. coli strains harboring barcoded plasmids that express a sgRNA which suppress different genes through dCas9.

Proceedings of the National Academy of Sciences, 2017

Significance Antibiotic resistance is a global threat to human health. The problem is aggravated ... more Significance Antibiotic resistance is a global threat to human health. The problem is aggravated by unnecessary and incorrect use of broad spectrum antibiotics. One way to provide correct treatment and slow down the development of antibiotic resistance is to assay the susceptibility profile of the infecting bacteria before treatment is initiated and let this information guide the choice of antibiotic. Here, we present an antibiotic susceptibility test that is sufficiently fast to be used at the point of care. We show that it is possible to determine if a urinary tract infection is caused by resistant bacteria within 30 min of loading a urine sample, even if the bacterial concentration in the urine is very low.

The emergence and spread of antibiotic resistant bacteria is a global threat to human health. The... more The emergence and spread of antibiotic resistant bacteria is a global threat to human health. The problem is aggravated by unnecessary and incorrect use of broad-spectrum antibiotics. One way to provide correct treatment and to slow down the development of antibiotic resistance is to assay the susceptibility profile of the infecting bacteria before treatment is initiated and let this information guide the choice of antibiotic. However, current methods for Antibiotics Susceptibility Testing (AST) are too slow for point of care application. Here we present a fast AST, fASTest, that rapidly captures individual bacterial cells in nanofluidics channels and monitors their response to different antibiotics based on direct imaging. By averaging the growth rate over many cells, we determined the susceptibility to several antibiotics in less than 25 min even at cell densities as low as 10000 CFU/mL. The short time scale, high sensitivity and high specificity make the method practically useful...

Cell, 2016

Highlights d Replication initiates at a nearly fixed volume per chromosome for all growth rates d... more Highlights d Replication initiates at a nearly fixed volume per chromosome for all growth rates d The time from initiation to division depends on the individual cell's growth rate d Variation in growth rate sets the variation in generation time and division size d E. coli appears as a ''sizer'' at slow growth and an ''adder'' at fast growth

Journal of Veterinary Pharmacology and Therapeutics, 2018

VetCAST is the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee f... more VetCAST is the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for Veterinary Antimicrobial Susceptibility Testing. Its remit is to define clinical breakpoints (CBPs) for antimicrobial drugs (AMDs) used in veterinary medicine in Europe. This position paper outlines the procedures and reviews scientific options to solve challenges for the determination of specific CBPs for animal species, drug substances and disease conditions. VetCAST will adopt EUCAST approaches: the initial step will be data assessment; then procedures for decisions on the CBPs; and finally the release of recommendations for CBP implementation. The princi

Blood stream infection (BSI) is related to high mortality and morbidity. Early antimicrobial ther... more Blood stream infection (BSI) is related to high mortality and morbidity. Early antimicrobial therapy is crucial in treating patients with BSI. The most common Gram-negative bacteria causing BSI is Escherichia coli. Targeted effective treatment of patients with BSI is only possible if it is based on antibiotic susceptibility testing (AST) data after blood culture positivity. However, there are very few methods available for rapid phenotypic AST and the fastest method takes 4 h. Here we analyzed the performance of a 30 min ultra-rapid method for AST of E. coli directly from positive blood cultures (BC). In total, 51 positive BC with E. coli were studied, and we evaluated the ultra-rapid method directly on positive BC as well as on E. coli colonies cultured on agar plates. The results obtained by the new method were compared with disk diffusion. The method provided accurate AST result in 30 min to Ciprofloxacin and Gentamicin for 92% and 84% of the positive BC samples, respectively. Fo...

arXiv: Quantitative Methods, 2015

Isogenic Escherichia coli growing exponentially in a constant environment display large variation... more Isogenic Escherichia coli growing exponentially in a constant environment display large variation in growth-rates, division-sizes and generation-times. It is unclear how these seemingly random cell cycles can be reconciled with the precise regulation required under conditions where the generation time is shorter than the time to replicate the genome. Here we use single molecule microscopy to map the location of the replication machinery to the division cycle of individual cells. We find that the cell-to-cell variation in growth rate is sufficient to explain the corresponding variation in cell size and division timing assuming a simple mechanistic model. In the model, initiation of chromosome replication is triggered at a fixed volume per origin region, and associated with each initiation event is a division event at a growth rate dependent time later. The result implies that cell division in E. coli has no other regulation beyond what is needed to initiate DNA replication at the rig...

Gene regulation mediated by transcription factors (TFs) is essential for all organisms. The funct... more Gene regulation mediated by transcription factors (TFs) is essential for all organisms. The functionality of TFs can largely be described by the fraction of time they occupy their regulatory binding sites on the chromosome. DNA-binding proteins have been shown to find their targets through facilitated diffusion in vitro. In its simplest form this means that the protein combines a random 3D search in the cytoplasm with 1D sliding along DNA. This has been proposed to speed up target location. It is difficult to mimic the in vivo conditions for gene regulation in biochemistry experiments; i.e. the ionic strength, chromosomal structure, and the presence of other DNA-binding macromolecules. In this thesis single molecule imaging assays for live cell measurements were developed to study the kinetics of the Escherichia coli transcription factor LacI. The low copy number LacI, in fusion with a fluorescent protein (Venus) is detected as a localized near-diffraction limited spot when being DN...

The EMBO journal, Jan 2, 2018

Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. ... more Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing...

So far, it has not been possible to perform advanced microscopy on pool generated strain librarie... more So far, it has not been possible to perform advanced microscopy on pool generated strain libraries and at the same time know each strain’s genotype. We have overcome this barrier by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single molecule fluorescence imaging of E. coli strains harboring barcoded plasmids that express a sgRNA which suppress different genes through dCas9.

Proceedings of the National Academy of Sciences, 2017

Significance Antibiotic resistance is a global threat to human health. The problem is aggravated ... more Significance Antibiotic resistance is a global threat to human health. The problem is aggravated by unnecessary and incorrect use of broad spectrum antibiotics. One way to provide correct treatment and slow down the development of antibiotic resistance is to assay the susceptibility profile of the infecting bacteria before treatment is initiated and let this information guide the choice of antibiotic. Here, we present an antibiotic susceptibility test that is sufficiently fast to be used at the point of care. We show that it is possible to determine if a urinary tract infection is caused by resistant bacteria within 30 min of loading a urine sample, even if the bacterial concentration in the urine is very low.

The emergence and spread of antibiotic resistant bacteria is a global threat to human health. The... more The emergence and spread of antibiotic resistant bacteria is a global threat to human health. The problem is aggravated by unnecessary and incorrect use of broad-spectrum antibiotics. One way to provide correct treatment and to slow down the development of antibiotic resistance is to assay the susceptibility profile of the infecting bacteria before treatment is initiated and let this information guide the choice of antibiotic. However, current methods for Antibiotics Susceptibility Testing (AST) are too slow for point of care application. Here we present a fast AST, fASTest, that rapidly captures individual bacterial cells in nanofluidics channels and monitors their response to different antibiotics based on direct imaging. By averaging the growth rate over many cells, we determined the susceptibility to several antibiotics in less than 25 min even at cell densities as low as 10000 CFU/mL. The short time scale, high sensitivity and high specificity make the method practically useful...

Cell, 2016

Highlights d Replication initiates at a nearly fixed volume per chromosome for all growth rates d... more Highlights d Replication initiates at a nearly fixed volume per chromosome for all growth rates d The time from initiation to division depends on the individual cell's growth rate d Variation in growth rate sets the variation in generation time and division size d E. coli appears as a ''sizer'' at slow growth and an ''adder'' at fast growth

Journal of Veterinary Pharmacology and Therapeutics, 2018

VetCAST is the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee f... more VetCAST is the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for Veterinary Antimicrobial Susceptibility Testing. Its remit is to define clinical breakpoints (CBPs) for antimicrobial drugs (AMDs) used in veterinary medicine in Europe. This position paper outlines the procedures and reviews scientific options to solve challenges for the determination of specific CBPs for animal species, drug substances and disease conditions. VetCAST will adopt EUCAST approaches: the initial step will be data assessment; then procedures for decisions on the CBPs; and finally the release of recommendations for CBP implementation. The princi