scholarly journals Candida glabrata MSH2 deletion increases antifungal tolerance in vitro and during intra-abdominal candidiasis (IAC), it does not impact pathogenesis of peritonitis or intra-abdominal abscesses

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Shaoji Cheng ◽  
Guojun Liu ◽  
Cornelius Joseph Clancy ◽  
Minh Hong Thi Nguyen
Keyword(s):  
Common Type ◽  
Dna Mismatch ◽  
Growth Defects ◽  
Abdominal Abscesses ◽  
Solid Media ◽  
Damage Response ◽  
Mutant Strains ◽  
Fluconazole Resistant ◽  
Resistant Mutants

Background: IAC is the second most common type of invasive Candidiasis, but its pathogenesis is poorly understood. We have shown that Candida albicans DNA damage response genes are strongly induced within intra-abdominal abscesses. Deletion of C. glabrata MSH2, A DNA mismatch repair (MMR) gene, results in a mutator phenotype that facilitates multidrug resistance in vitro and in mouse gastrointestinal tracts. Our goal was to determine if CGMSH2 Contributed to pathogenesis or resistance to the new antifungal rezafungin during IAC. Methods: We createdΔMSH2 in BG2 using SAT-Flipper, and tested virulence and rezafungin responses in a mouse model of IAC. Results: ΔMSH2 displayed no growth defects at 30°C in liquid (YPD, Ypglycerol) or solid media (YPD+0.02% MMS, 1MM H2O2, 1M NACL, 20 UG/ML CW, 250 UG/ML OR 0.02% SDS). ΔMSH2 longevity in YPD was comparable to BG2. Caspofungin-, Rezafungin- and Fluconazole-resistant mutants arose 24-, 16- and 3-fold more often, respectively, for ΔMSH2 than BG2 (108-106 CFU overnight in YPD, selected on 8XMIC-Containing plates). However, respective minimum inhibitory concentrations (MICS) were not different, nor were rezafungin time-kills.ΔMSH2 was comparable to BG2 in peritonitis and abscess burdens in mouse IAC.ΔMSH2 demonstrated significantly greater caspofungin- and fluconazole-tolerance than BG2 in abscesses. Rezafungin reduced peritonitis and abscess burdens ofΔMSH2,BG2 ANDFKS mutant strains to similar extents. Conclusions: CgMSH2 deletionincreased the frequency of spontaneously-arising echinocandin- and fluconazole-resistant colonies in vitro and tolerance in intra-abdominal abscesses, but it did not attenuate virulence or rezafungin responses during IAC.

scholarly journals The Ribosome-Bound Chaperones RAC and Ssb1/2p Are Required for Accurate Translation in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (20) ◽  
pp. 9186-9197 ◽  
Author(s):  
Magdalena Rakwalska ◽  
Sabine Rospert
Keyword(s):  
Translation Termination ◽  
Ribosomal Subunit ◽  
Reporter System ◽  
Translational Fidelity ◽  
Growth Defects ◽  
Mutant Strains ◽  
Translation Termination Factor ◽  
Combined Data

ABSTRACT The chaperone homologs RAC (ribosome-associated complex) and Ssb1/2p are anchored to ribosomes; Ssb1/2p directly interacts with nascent polypeptides. The absence of RAC or Ssb1/2p results in a similar set of phenotypes, including hypersensitivity against the aminoglycoside paromomycin, which binds to the small ribosomal subunit and compromises the fidelity of translation. In order to understand this phenomenon we measured the frequency of translation termination and misincorporation in vivo and in vitro with a novel reporter system. Translational fidelity was impaired in the absence of functional RAC or Ssb1/2p, and the effect was further enhanced by paromomycin. The mutant strains suffered primarily from a defect in translation termination, while misincorporation was compromised to a lesser extent. Consistently, a low level of soluble translation termination factor Sup35p enhanced growth defects in the mutant strains. Based on the combined data we conclude that RAC and Ssb1/2p are crucial in maintaining translational fidelity beyond their postulated role as chaperones for nascent polypeptides.

scholarly journals In Vitro Selection of Resistance to Clinafloxacin, Ciprofloxacin, and Trovafloxacin in Streptococcus pneumoniae

2000 ◽  
Vol 44 (10) ◽  
pp. 2740-2746 ◽  
Author(s):  
Kensuke Nagai ◽  
Todd A. Davies ◽  
Glenn A. Pankuch ◽  
Bonifacio E. Dewasse ◽  
Michael R. Jacobs ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Single Step ◽  
Mutation Rates ◽  
Mutant Strains ◽  
Efflux Mechanism ◽  
Resistant Mutants ◽  
Time Kill ◽  
Selection Of ◽  
Selection Of Resistance

ABSTRACT Ability of daily sequential subcultures in subinhibitory concentrations of clinafloxacin, ciprofloxacin, and trovafloxacin to select resistant mutants was studied in 10 pneumococci (ciprofloxacin MICs, 1 to 4 μg/ml, and clinafloxacin and trovafloxacin MICs, 0.06 to 0.125 μg/ml [n = 9]; ciprofloxacin, clinafloxacin, and trovafloxacin MICs, 32, 0.5, and 2 μg/ml, respectively [n = 1]). Subculturing was done 50 times, or until MICs increased fourfold or more. Mutants for which MICs were fourfold (or more) higher than those for parent strains were selected in five strains by clinafloxacin, in six strains by trovafloxacin, and nine strains by ciprofloxacin. Sequence analysis of type II topoisomerase showed that most mutants had mutations in ParC at Ser79 or Asp83 and in GyrA at Ser81, while a few mutants had mutations in ParE or GyrB. In the presence of reserpine, the MICs of ciprofloxacin and clinafloxacin for most mutants were lower (four to eight times lower), but for none of the mutants were trovafloxacin MICs lower, suggesting an efflux mechanism affecting the first two agents but not trovafloxacin. Single-step mutation rates were also determined for eight strains for which the MICs were as follows: 0.06 μg/ml (clinafloxacin), 0.06 to 0.125 μg/ml (trovafloxacin), and 1 μg/ml (ciprofloxacin). Single-step mutation rates with drugs at the MIC were 2.0×10−9 to <1.1×10−11, 5.0×10−4 to 3.6×10−9, and 4.8×10−4 to 6.7×10−9, respectively. For two strains with clinafloxacin MICs of 0.125 to 0.5 μg/ml trovafloxacin MICs of 0.125 to 2 μg/ml, ciprofloxacin MICs of 4 to 32 μg/ml mutation rates with drugs at the MIC were 1.1×10−8−9.6×10−8, 3.3×10−6−6.7×10−8, and 2.3×10−5−2.4×10−7, respectively. Clinafloxacin was bactericidal at four times the MIC after 24 h against three parent and nine mutant strains by time-kill study. This study showed that single and multistep clinafloxacin exposure selected for resistant mutants less frequently than similar exposures to other drugs studied.

scholarly journals Evolutionary repair reveals an unexpected role of the tRNA modification m1G37 in aminoacylation

2021 ◽  
Author(s):  
Ben E Clifton ◽  
Muhammad Aiman Fariz ◽  
Gen-Ichiro Uechi ◽  
Paola Laurino
Keyword(s):  
Experimental Evolution ◽  
Protein Translation ◽  
Trna Modification ◽  
E Coli ◽  
Growth Defects ◽  
Translational Accuracy ◽  
Translational Frameshift ◽  
Mutant Strains ◽  
Trna Methyltransferase

The tRNA modification m1G37, which is introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria due to its role in suppressing translational frameshift errors at proline codons. However, because bacteria can tolerate high levels of mistranslation, it is unclear why loss of m1G37 is not tolerated. Here, we addressed this question by performing experimental evolution of trmD mutant strains of E. coli. Surprisingly, trmD mutant strains were viable even if the m1G37 modification was completely abolished, and showed rapid recovery of growth rate, mainly via tandem duplication or coding mutations in the proline-tRNA ligase gene proS. Growth assays and in vitro aminoacylation assays showed that G37-unmodified tRNAPro is aminoacylated less efficiently than m1G37-modified tRNAPro, and that growth of trmD mutant strains can be largely restored by single mutations in proS that restore aminoacylation of G37-unmodified tRNAPro. These results show that inefficient aminoacylation of tRNAPro is the main reason for growth defects observed in trmD mutant strains and that the ProRS enzyme may act as a gatekeeper of translational accuracy, preventing the use of error-prone unmodified tRNAPro in protein translation. Our work shows the utility of experimental evolution for uncovering the hidden functions of essential genes and has implications for the development of antibiotics targeting TrmD.

scholarly journals Mutations in fbiD (Rv2983) as a novel determinant of resistance to pretomanid and delamanid in Mycobacterium tuberculosis

2020 ◽  
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Solid Media ◽  
Resistant Mutants ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole pro-drugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 91% of which occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance: fbiC (56%), fbiA (15%), ddn (12%), fgd (4%) and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983, a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance, but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Sequencing of Gyrase and Topoisomerase IV Quinolone-Resistance-Determining Regions of Chlamydia trachomatis and Characterization of Quinolone-Resistant Mutants Obtained In Vitro

1998 ◽  
Vol 42 (10) ◽  
pp. 2474-2481 ◽  
Author(s):  
Sophie Dessus-Babus ◽  
Cécile M. Bébéar ◽  
Alain Charron ◽  
Christiane Bébéar ◽  
Bertille de Barbeyrac
Keyword(s):  
Chlamydia Trachomatis ◽  
Reference Strain ◽  
Fold Increase ◽  
Quinolone Resistance ◽  
Topoisomerase Iv ◽  
Mutant Strains ◽  
Resistant Strains ◽  
Resistant Mutants ◽  
High Level

ABSTRACT The L2 reference strain of Chlamydia trachomatis was exposed to subinhibitory concentrations of ofloxacin (0.5 μg/ml) and sparfloxacin (0.015 μg/ml) to select fluoroquinolone-resistant mutants. In this study, two resistant strains were isolated after four rounds of selection. The C. trachomatis mutants presented with high-level resistance to various fluoroquinolones, particularly to sparfloxacin, for which a 1,000-fold increase in the MICs for the mutant strains compared to the MIC for the susceptible strain was found. The MICs of unrelated antibiotics (doxycycline and erythromycin) for the mutant strains were identical to those for the reference strain. The gyrase (gyrA, gyrB) and topoisomerase IV (parC, parE) genes of the susceptible and resistant strains of C. trachomatis were partially sequenced. A point mutation was found in the gyrAquinolone-resistance-determining region (QRDR) of both resistant strains, leading to a Ser83→Ile substitution (Escherichia coli numbering) in the corresponding protein. ThegyrB, parC, and parE QRDRs of the resistant strains were identical to those of the reference strain. These results suggest that in C. trachomatis, DNA gyrase is the primary target of ofloxacin and sparfloxacin.

scholarly journals Resistance Studies with Daptomycin

2001 ◽  
Vol 45 (6) ◽  
pp. 1799-1802 ◽  
Author(s):  
Jared A. Silverman ◽  
Nicole Oliver ◽  
Ted Andrew ◽  
Tongchuan Li
Keyword(s):  
Population Analysis ◽  
Serial Passage ◽  
Chemical Mutagenesis ◽  
Growth Defects ◽  
Drug Concentrations ◽  
Small Colony ◽  
Resistant Mutants ◽  
Resistance Rates

ABSTRACT We studied the in vitro emergence of resistance to daptomycin using three methods: spontaneous resistance incidence, serial passage in the presence of increasing drug concentrations, and chemical mutagenesis. No spontaneously resistant mutants were obtained for any organism tested (<10−10 for Staphylococcus aureus, <10−9 for Staphylococcus epidermidis, <10−9 for Enterococcus faecalis, <10−9 for Enterococcus faecium, <10−8 for Streptococcus pneumoniae). Population analysis demonstrated that bacterial susceptibility to daptomycin is heterogeneous. Assay results were sensitive to calcium concentration and culture density, both of which can affect apparent resistance rates. Stable S. aureus mutants were isolated by both serial passage in liquid media and chemical mutagenesis. The daptomycin MICs for these isolates were 8- to 32-fold higher than for the parental strain. Many mutants with high MICs (>12.5 μg/ml) had significant growth defects but did not display phenotypes typical ofS. aureus small colony variants. The voltage component (Δψ) of the bacterial membrane potential was increased in three independent resistant isolates. In vivo data showed that some daptomycin-resistant mutants had lost significant virulence. For other mutants, the degree of in vitro resistance was greater than the change in in vivo susceptibility. These results suggest that infection with some daptomycin-resistant organisms may still be easily treatable.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

Characterization of sparfloxacin-resistant mutants of Staphylococcus aureus obtained in vitro

2001 ◽  
Vol 18 (2) ◽  
pp. 107-112 ◽  
Author(s):  
Joaquim Ruiz ◽  
Josep M. Sierra ◽  
M.Teresa Jiménez De Anta ◽  
Jordi Vila
Keyword(s):  
Staphylococcus Aureus ◽  
Resistant Mutants

scholarly journals Evaluation of Antimicrobial, Antioxidant, and Cytotoxic Activity of Phenolic Preparations of Diverse Composition, Obtained from Elaeagnus rhamnoides (L.) A. Nelson Leaf and Twig Extracts

Molecules ◽  
2021 ◽  
Vol 26 (10) ◽  
pp. 2835
Author(s):  
Anna Stochmal ◽  
Bartosz Skalski ◽  
Rostyslav Pietukhov ◽  
Beata Sadowska ◽  
Joanna Rywaniak ◽  
...  
Keyword(s):  
Chemical Composition ◽  
Plasma Lipids ◽  
Biological Properties ◽  
Sea Buckthorn ◽  
Phenolic Antioxidants ◽  
Microdilution Method ◽  
Solid Media ◽  
Broth Microdilution Method ◽  
Markers Of Oxidative Stress

Although the major components of various organs of sea buckthorn have been identified (particularly phenolic compounds), biological properties of many of these phytochemicals still remain poorly characterized. In this study, we focused on the chemical composition and biological activity of preparations that were obtained from sea buckthorn twigs and leaves. The objective was to investigate cytotoxicity of these preparations against human fibroblast line HFF-1, using MTT reduction assay, their anti- or pro-oxidant activities against the effects of a biological oxidant -H2O2/Fe—on human plasma lipids and proteins in vitro (using TBARS and carbonyl groups as the markers of oxidative stress). Antimicrobial activity of the tested preparations against Gram-positive (Staphylococcus aureus, S. epidermidis, Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), as well as against fungi (Candida albicans, C. glabrata) by the EUCAST-approved broth microdilution method, followed by growth on solid media, were also assessed. Our analysis showed significant differences in chemical composition and biological properties of the tested preparations (A–F). All tested preparations from sea buckthorn twigs (D–F) and one preparation from sea buckthorn leaves (preparation C) may be a new source of phenolic antioxidants for pharmacological and cosmetic applications.

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