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1.
mBio ; 15(2): e0282823, 2024 Feb 14.
Article in English | MEDLINE | ID: mdl-38193698

ABSTRACT

Streptococcus pneumoniae is a major human pathogen of global health concern and the rapid emergence of antibiotic resistance poses a serious public health problem worldwide. Fluoroquinolone resistance in S. pneumoniae is an intriguing case because the prevalence of fluoroquinolone resistance does not correlate with increasing usage and has remained rare. Our data indicate that deleterious fitness costs in the mammalian host constrain the emergence of fluoroquinolone resistance both by de novo mutation and recombination. S. pneumoniae was able to circumvent such deleterious fitness costs via the development of antibiotic tolerance through metabolic adaptation that reduced the production of reactive oxygen species, resulting in a fitness benefit during infection of mice treated with fluoroquinolones. These data suggest that the emergence of fluoroquinolone resistance is tightly constrained in S. pneumoniae by fitness tradeoffs and that mutational pathways involving metabolic networks to enable tolerance phenotypes are an important contributor to the evasion of antibiotic-mediated killing.IMPORTANCEThe increasing prevalence of antibiotic resistant bacteria is a major global health concern. While many species have the potential to develop antibiotic resistance, understanding the barriers to resistance emergence in the clinic remains poorly understood. A prime example of this is fluroquinolone resistance in Streptococcus pneumoniae, whereby, despite continued utilization, resistance to this class of antibiotic remains rare. In this study, we found that the predominant pathways for developing resistance to this antibiotic class severely compromised the infectious capacity of the pneumococcus, providing a key impediment for the emergence of resistance. Using in vivo models of experimental evolution, we found that S. pneumoniae responds to repeated fluoroquinolone exposure by modulating key metabolic pathways involved in the generation of redox molecules, which leads to antibiotic treatment failure in the absence of appreciable shifts in resistance levels. These data underscore the complex pathways available to pathogens to evade antibiotic mediating killing via antibiotic tolerance.


Subject(s)
Fluoroquinolones , Pneumococcal Infections , Humans , Animals , Mice , Fluoroquinolones/pharmacology , Streptococcus pneumoniae/metabolism , Pneumococcal Infections/drug therapy , Pneumococcal Infections/microbiology , Drug Resistance, Bacterial/genetics , Microbial Sensitivity Tests , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolism , Mammals
2.
J Med Microbiol ; 71(12)2022 Dec.
Article in English | MEDLINE | ID: mdl-36748534

ABSTRACT

This study evaluated the HiberGene Group B Streptococcus test, a CE-IVD-approved molecular assay for rapid detection of Streptococcus agalactiae [Group B Streptococcus (GBS)] in human clinical specimens. Performance of the assay in terms of specificity, sensitivity and genotype inclusivity was investigated using an extended specificity panel of 113 human and animal GBS isolates, and eight isolates from other streptococcal species, from the isolate collection of the German National Reference Center for Streptococci. Broth cultures were tested according to the manufacturer's protocol, including lysis, heat denaturation and isothermal amplification. All 104/104 (100 %) human GBS isolates of nine serotypes (Ia, Ib, II, III, IV, V, VI, VII, VIII and non-typeable) were correctly identified by the assay as GBS. Additionally, 7/9 (78 %) GBS isolates from elephants were also correctly identified. Six isolates of other streptococcal species/subspecies (Streptococcus anginosus, S. constellatus, S. castoreus, S. dysgalactiae and S. dysgalactiae equisimilis) were correctly reported as negative. Two S. pyogenes (Group A Streptococcus) isolates gave invalid results. The HG Group B Streptococcus assay identified human GBS isolates in culture with 100 % sensitivity.


Subject(s)
Streptococcal Infections , Streptococcus agalactiae , Animals , Humans , Streptococcal Infections/diagnosis , Streptococcal Infections/veterinary , Nucleic Acid Amplification Techniques/methods , Streptococcus pyogenes/genetics
3.
J Steroid Biochem Mol Biol ; 211: 105880, 2021 07.
Article in English | MEDLINE | ID: mdl-33757894

ABSTRACT

Steroids play an important role in cell regulation and homeostasis. Many diseases like Alzheimer's disease or Smith-Lemli-Opitz syndrome are known to be associated with deviations in the steroid profile. Most published methods only allow the analysis of small subgroups of steroids and cannot give an overview of the total steroid profile. We developed and validated a method that allows the analysis of free neutral steroids, including intermediates of cholesterol biosynthesis, free oxysterols, C19 and C21 steroids, free steroid acids, including bile acids, and sterol sulfates using gas chromatography-mass spectrometry. Samples were analyzed in scan mode for screening purposes and in dynamic multiple reaction monitoring mode for highly sensitive quantitative analysis. The method was validated for mouse brain and liver tissue and consists of sample homogenization, lipid extraction, steroid group separation, deconjugation, derivatization and gas chromatography-mass spectrometry analysis. We applied the method on brain and liver samples of mice (10 months and 3 weeks old) and cultured N2a cells and report the endogenous concentrations of 29 physiological steroids.


Subject(s)
Brain/metabolism , Gas Chromatography-Mass Spectrometry/methods , Liver/metabolism , Steroids/analysis , Sulfates/analysis , Animals , Female , Male , Mice , Mice, Inbred C57BL , Steroids/metabolism , Sulfates/metabolism
4.
Nat Protoc ; 14(8): 2546-2570, 2019 08.
Article in English | MEDLINE | ID: mdl-31341291

ABSTRACT

Distal cholesterol biosynthesis (CB) has recently taken center stage as a promising drug target in several diseases previously not linked to this biochemical pathway, including cardiovascular disease, cancer, multiple sclerosis and Alzheimer's disease. Most enzymes involved in this pathway are hard to isolate, warranting dedicated analytical tools for biochemical screening. We describe the use of gas chromatography-electron ionization mass spectrometry (GC-MS) in a whole-cell screening assay aimed at monitoring interactions with all enzymes of distal CB in a single experiment. Following cell culture and lipid extraction, the trimethylsilyl ethers of sterols are analyzed by GC-MS. Analytical data for 23 relevant sterols (intermediates) are provided, allowing their unambiguous identification. Sterol pattern analysis reveals the target enzyme on the basis of characteristic marker sterols, whereas quantification of 2-13C-acetate incorporation correlates with the inhibitory activity of drug candidates. The protocol can be used by both experienced scientists and newcomers to the field, allowing detection and quantification of small molecule-enzyme interactions in distal CB. The entire protocol can be carried out within two working days.


Subject(s)
Cholesterol/metabolism , Cytological Techniques/methods , Gas Chromatography-Mass Spectrometry/methods , Biological Assay/methods , Cholesterol/analysis , Cholesterol/chemistry , Drug Delivery Systems , HL-60 Cells , Humans , Sterols/analysis , Sterols/chemistry , Sterols/metabolism
5.
Molecules ; 24(13)2019 Jun 26.
Article in English | MEDLINE | ID: mdl-31247920

ABSTRACT

Sulfoconjugates of sterols play important roles as neurosteroids, neurotransmitters, and ion channel ligands in health and disease. In most cases, sterol conjugate analysis is performed with liquid chromatography-mass spectrometry. This is a valuable tool for routine analytics with the advantage of direct sterol sulfates analysis without previous cleavage and/or derivatization. The complementary technique gas chromatography-mass spectrometry (GC-MS) is a preeminent discovery tool in the field of sterolomics, but the analysis of sterol sulfates is hampered by mandatory deconjugation and derivatization. Despite the difficulties in sample workup, GC-MS is an indispensable tool for untargeted analysis and steroid profiling. There are no general sample preparation protocols for sterol sulfate analysis using GC-MS. In this study we present a reinvestigation and evaluation of different deconjugation and derivatization procedures with a set of representative sterol sulfates. The advantages and disadvantages of trimethylsilyl (TMS), methyloxime-trimethylsilyl (MO-TMS), and trifluoroacetyl (TFA) derivatives were examined. Different published procedures of sterol sulfate deconjugation, including enzymatic and chemical cleavage, were reinvestigated and examined for diverse sterol sulfates. Finally, we present a new protocol for the chemical cleavage of sterol sulfates, allowing for simultaneous deconjugation and derivatization, simplifying GC-MS based sterol sulfate analysis.


Subject(s)
Gas Chromatography-Mass Spectrometry , Steroids/chemistry , Sterols/chemistry , Sulfates/chemistry , Humans , Molecular Structure , Solvents , Steroids/analysis , Sterols/analysis , Sulfates/analysis
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