scholarly journals Identification of the Genetic Basis for Clinical Menadione-Auxotrophic Small-Colony Variant Isolates of Staphylococcus aureus

2008 ◽  
Vol 52 (11) ◽  
pp. 4017-4022 ◽  
Author(s):  
Jonas Lannergård ◽  
Christof von Eiff ◽  
Gunnar Sander ◽  
Tina Cordes ◽  
Jochen Seggewiβ ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Therapy ◽  
Hemolytic Activity ◽  
Genetic Basis ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Normal Growth ◽  
Gene Encoding ◽  
Systemic Antibiotic Therapy ◽  
Small Colony

ABSTRACT Small-colony variants (SCVs) of Staphylococcus aureus are associated with persistent infections and may be selectively enriched during antibiotic therapy. Three pairs of clonally related S. aureus isolates were recovered from patients receiving systemic antibiotic therapy. Each pair consisted of an isolate with a normal phenotype and an isolate with an SCV phenotype. These SCVs were characterized by reduced susceptibility to gentamicin, reduced hemolytic activity, slow growth, and menadione auxotrophy. Sequencing of the genes involved in menadione biosynthesis revealed mutations in menB, the gene encoding naphthoate synthase, in all three strains with the SCV phenotype. The menB mutations were (i) a 9-bp deletion from nucleotides 55 to 63, (ii) a frameshift mutation that resulted in a premature stop codon at position 230, and (iii) a point mutation that caused the amino acid substitution Gly to Val at codon 233. Fluctuation tests showed that growth-compensated mutants arose in the SCV population of one strain, strain OM1b, at a rate of 1.8 × 10−8 per cell per generation. Sequence analyses of 23 independently isolated growth-compensated mutants of this strain revealed alterations in the menB sequence in every case. These alterations included reversions to the wild-type sequence and intragenic second-site mutations. Each of the growth-compensated mutants showed a restoration of normal growth and a loss of menadione auxotrophy, increased susceptibility to gentamicin, and restored hemolytic activity. These data show that mutations in menB cause the SCV phenotype in these clinical isolates. This is the first report on the genetic basis of menadione-auxotrophic SCVs determined in clinical S. aureus isolates.

scholarly journals Use of Staphylococcus aureus Phage Lysate Staphage Lysate (SPL)® for the Control of Recurrent Pyoderma Eczema in Dogs with Atopic Dermatitis

2018 ◽  
Vol 44 (1) ◽  
pp. 7 ◽  
Author(s):  
Suzana Evelyn Bahr Solomon ◽  
Marconi Rodrigues de Farias ◽  
Claudia Turra Pimpão
Keyword(s):  
Staphylococcus Aureus ◽  
Atopic Dermatitis ◽  
Antibiotic Therapy ◽  
Significant Decline ◽  
Microbiological Analysis ◽  
Phage Lysate ◽  
Systemic Antibiotic ◽  
Therapeutic Protocol ◽  
Systemic Antibiotic Therapy ◽  
The Mean

Background: Recurrent staphylococcal infections are frequent in dogs with atopic dermatitis (AD). Many factors seem to contribute to making bacterial pyoderma refractory to treatment. Short-term systemic antibiotic therapy is effective for the treatment of acute symptoms, and may, along with pulsatile therapy, contribute to the long-term control of the disease. However, microbial resistance has become a growing and alarming problem. The aim of this study was to evaluate whether the use of Staphylococcus aureus Phage Lysate Staphage Lysate (SPL)®, can minimize the symptoms of recurrent pyoderma and increase the interval between acute atopic manifestations in dogs.Materials, Methods & Results: Thirteen dogs with a history of Canine Atopic Dematitis (CAD) and recurrent bacterial pyoderma received SPL at increasing intervals for 23 weeks. The contents of an intact pustule of each dog was collected and submitted to microbiological analysis. Systemic antibiotic therapy was established for the first 4-6 weeks of SPL protocol, based on the antibiotic sensitivity tests. The animals included in the study underwent a therapeutic protocol receiving shots of 0.5 mL of SPL subcutaneously (SC) twice a week for the first 12 weeks; 1.0 mL of SPL (SC) once a week for four weeks; 1.0 mL of SPL (SC) once every 15 days; 1.0 mL of SPL (SC) after a three-week interval from the last dose on week 20, until final observation at week 26, with no application. The animals underwent clinical examination every week and the evaluation of pruritus was used according Rybnicek et al. During the therapeutic protocol with SPL, a significant decline in the pruritus was observed in the treated dogs (P < 0.05). In week 1, the mean pruritus index was 7.33 on the Rybnicek scale; in weeks 12 and 23, the mean indices were 2.41 and 1.91. An effectiveness of 83.33% for the control of pruritus along with regression of the lesions was observed.Discussion: Before treatment, the selected animals presented worsening of the pruritus during the pyoderma eczema episodes (pruritic), resulting in the emergence of a vicious cycle where the pruritus induced the appearance of new lesions, requiring the use of antibiotics for a long period. During the therapeutic protocol with SPL, a significant decline in the pruritus was observed in the treated dogs. The control of pruritus associated with pyoderma eczema of the dogs in this study before the vaccination protocol with SPL was satisfactory when they were subjected to antibiotic therapy; however, after suspending therapy, the bacterial infections recurred, on average, after 2-4 weeks. On the other hand, with the use of SPL, the animals were recurrence-free until the end of the experimental protocol. This was attributed to the antibiotic therapy administered at the beginning of the protocol, as this led to a regression of the bacterial pyoderma and involution of the lesions. However, after suspending antibiotics, it was observed that, by the end of the study, 83.33% of the dogs still had a low level of pruritus, few or no lesions, which were considered acceptable to most owners. At this moment none of these patients needed to be subjected to antibiotic treatment. The sums of the scores for the dogs on weeks 1, 12, and 23 were 53.33, 4.41, and 3.5, respectively, indicating significant improvements of the lesions, showing that the proposed protocol with SPL was able to prevent new episodes of pyoderma.

scholarly journals SAT-286 TSH Synthesis and Secretion Are Unperturbed in Male IRS4 Knockout Mice

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Emilie Brule ◽  
Anita Boelen ◽  
Daniel J Bernard
Keyword(s):  
Knockout Mice ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Whole Body ◽  
Gene Encoding ◽  
Central Hypothyroidism ◽  
Guide Rna ◽  
Downstream Effectors ◽  
Tsh Secretion ◽  
Hpt Axis

Abstract It was recently reported that mutations in the insulin receptor substrate 4 (IRS4) gene cause a novel form of X-linked congenital central hypothyroidism (OMIM 300904). To date, four different mutations, three frameshift and one nonsense, have been reported, with two affected male patients showing decreased basal, pulsatile, and total thyroid-stimulation hormone (TSH) secretion (PMID 30061370). Members of the IRS family canonically act as scaffold proteins between tyrosine kinase receptors and their downstream effectors. IRS4/Irs4 expression is enriched in the pituitary; however, its role in the hypothalamic-pituitary-thyroid (HPT) axis has not been studied in detail. We generated novel whole-body Irs4-knockout mouse lines using CRISPR-Cas9. A specific guide RNA was used to target the Cas9 enzyme to the 5’ end of the single exon Irs4 gene. A two-nucleotide deletion was introduced into Irs4, resulting in a frameshift and premature stop codon. We hypothesized that like IRS4 deficient patients, these mice would exhibit central hypothyroidism. Given that Irs4 is X-linked, we focused our initial characterization on males. Under normal laboratory conditions, Irs4 knockout mice do not exhibit differences in pituitary expression of Tshb, which encodes one of the subunits of the TSH heterodimer. Expression of the gene encoding the thyrotropin-releasing hormone (TRH) receptor, Trhr1, is also unperturbed in these knockout mice. Additionally, there are no differences in their serum thyroid hormones, T3 (triiodothyronine) and T4 (thyroxine). When Irs4 knockout males were placed on a low-iodine diet supplemented with propylthiouracil (PTU) for 3 weeks and rendered hypothyroid, their serum TSH increased similarly to wild-type males. Overall, Irs4 knockout males do not exhibit central hypothyroidism or phenocopy IRS4 deficient patients. Compensation by another IRS protein may explain euthyroidism in these mice.

scholarly journals Unstable chromosome rearrangements in Staphylococcus aureus cause phenotype switching associated with persistent infections

2019 ◽  
Vol 116 (40) ◽  
pp. 20135-20140 ◽  
Author(s):  
Romain Guérillot ◽  
Xenia Kostoulias ◽  
Liam Donovan ◽  
Lucy Li ◽  
Glen P. Carter ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Genetic Basis ◽  
Host Cells ◽  
Type I ◽  
Modification System ◽  
Persistent Infections ◽  
Restriction Modification System ◽  
Long Read ◽  
Small Colony ◽  
Restriction Modification

Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type–like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.

scholarly journals Genetic Determinants of Resistance to Fusidic Acid among Clinical Bacteremia Isolates of Staphylococcus aureus

2009 ◽  
Vol 53 (5) ◽  
pp. 2059-2065 ◽  
Author(s):  
Jonas Lannergård ◽  
Tobias Norström ◽  
Diarmaid Hughes
Keyword(s):  
Staphylococcus Aureus ◽  
Fusidic Acid ◽  
Drug Target ◽  
Elongation Factor ◽  
Acid Resistance ◽  
Normal Growth ◽  
Genetic Determinants ◽  
Small Colony ◽  
Phylogenetic Lineages

ABSTRACT Resistance to fusidic acid in Staphylococcus aureus is caused by mutation of the elongation factor G (EF-G) drug target (FusA class) or by expression of a protein that protects the drug target (FusB and FusC classes). Recently, two novel genetic classes of small-colony variants (SCVs) were identified among fusidic acid-resistant mutants selected in vitro (FusA-SCV and FusE classes). We analyzed a phylogenetically diverse collection of fusidic acid-resistant bacteremia isolates to determine which resistance classes were prevalent and whether these were associated with particular phylogenetic lineages. Each isolate was shown by DNA sequencing and plasmid curing to carry only one determinant of fusidic acid resistance, with approximately equal frequencies of the FusA, FusB, and FusC genetic classes. The FusA class (mutations in fusA) were distributed among different phylogenetic types. Two distinct variants of the FusC class (chromosomal fusC gene) were identified, and FusC was also distributed among different phylogenetic types. In contrast, the FusB class (carrying fusB on a plasmid) was found in closely related types. No FusE-class mutants (carrying mutations in rplF) were found. However, one FusA-class isolate had multiple mutations in the fusA gene, including one altering a codon associated with the FusA-SCV class. SCVs are frequently unstable and may undergo compensatory evolution to a normal growth phenotype after their initial occurrence. Accordingly, this normal-growth isolate might have evolved from a fusidic acid-resistant SCV. We conclude that at least three different resistance classes are prevalent among fusidic acid-resistant bacteremia isolates of S. aureus.

scholarly journals Identification and characteristics of the structural gene for the Drosophila eye colour mutant sepia, encoding PDA synthase, a member of the Omega class glutathione S-transferases

2006 ◽  
Vol 398 (3) ◽  
pp. 451-460 ◽  
Author(s):  
Jaekwang Kim ◽  
Hyunsuk Suh ◽  
Songhee Kim ◽  
Kiyoung Kim ◽  
Chiyoung Ahn ◽  
...  
Keyword(s):  
Structural Gene ◽  
Stop Codon ◽  
Gel Filtration ◽  
Premature Stop Codon ◽  
Genomic Region ◽  
Gene Encoding ◽  
Colour Mutant ◽  
Eye Colour ◽  
Glutathione S Transferases

The eye colour mutant sepia (se1) is defective in PDA {6-acetyl-2-amino-3,7,8,9-tetrahydro-4H-pyrimido[4,5-b]-[1,4]diazepin-4-one or pyrimidodiazepine} synthase involved in the conversion of 6-PTP (2-amino-4-oxo-6-pyruvoyl-5,6,7,8-tetrahydropteridine; also known as 6-pyruvoyltetrahydropterin) into PDA, a key intermediate in drosopterin biosynthesis. However, the identity of the gene encoding this enzyme, as well as its molecular properties, have not yet been established. Here, we identify and characterize the gene encoding PDA synthase and show that it is the structural gene for sepia. Based on previously reported information [Wiederrecht, Paton and Brown (1984) J. Biol. Chem. 259, 2195–2200; Wiederrecht and Brown (1984) J. Biol. Chem. 259, 14121–14127; Andres (1945) Drosoph. Inf. Serv. 19, 45; Ingham, Pinchin, Howard and Ish-Horowicz (1985) Genetics 111, 463–486; Howard, Ingham and Rushlow (1988) Genes Dev. 2, 1037–1046], we isolated five candidate genes predicted to encode GSTs (glutathione S-transferases) from the presumed sepia locus (region 66D5 on chromosome 3L). All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro. The molecular mass of recombinant CG6781 was estimated to be 28 kDa by SDS/PAGE and 56 kDa by gel filtration, indicating that it is a homodimer under native conditions. Sequencing of the genomic region spanning CG6781 revealed that the se1 allele has a frameshift mutation from ‘AAGAA’ to ‘GTG’ at nt 190–194, and that this generates a premature stop codon. Expression of the CG6781 open reading frame in an se1 background rescued the eye colour defect as well as PDA synthase activity and drosopterins content. The extent of rescue was dependent on the dosage of transgenic CG6781. In conclusion, we have discovered a new catalytic activity for an Omega class GST and that CG6781 is the structural gene for sepia which encodes PDA synthase.

scholarly journals Growth requirements of some small-colony-forming variants of Staphylococcus aureus

1976 ◽  
Vol 4 (4) ◽  
pp. 343-348
Author(s):  
M L Kaplan ◽  
W Dye
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Unsaturated Fatty Acids ◽  
Normal Growth ◽  
Laboratory Procedure ◽  
Clinical Specimens ◽  
Growth Requirements ◽  
Small Colony ◽  
Metabolic Activities ◽  
Long Chain

Nine stable, auxotrophic, small-colony-forming variants of Staphylococcus aureus were isolated. They were of two types. One type, from broth cultures containing varying concentrations of gentamicin or kanamycin, required heme for normal growth. The second type, from the blood culture of a patient who had been treated with penicillin and oxacillin, required long-chain unsaturated fatty acids for normal growth. These small-colony-forming variants differed from their parents with respect to specific metabolic activities, many of which may be attributed to an impaired electron transport system and related functions. A laboratory procedure for detecting possible small-colony variants in clinical specimens was proposed.

scholarly journals AKNA Frameshift Variant in Three Dogs with Recurrent Inflammatory Pulmonary Disease

Genes ◽  
2019 ◽  
Vol 10 (8) ◽  
pp. 567 ◽  
Author(s):  
Hug ◽  
Anderegg ◽  
Kehl ◽  
Jagannathan ◽  
Leeb
Keyword(s):  
Pulmonary Disease ◽  
Clinical Symptoms ◽  
Stop Codon ◽  
Family Members ◽  
Premature Stop Codon ◽  
Homozygosity Mapping ◽  
Pedigree Analysis ◽  
Gene Encoding ◽  
Reading Frame ◽  
Inflammatory Processes

We investigated three related Rough Collies with recurrent inflammatory pulmonary disease. The clinical symptoms were similar to primary ciliary dyskinesia (PCD). However, the affected dogs did not carry any known pathogenic PCD variants. Pedigree analysis suggested a recessive mode of inheritance. Combined linkage and homozygosity mapping in three cases and seven non-affected family members delineated 19 critical intervals on 10 chromosomes comprising a total of 99 Mb. The genome of one affected dog was sequenced and compared to 601 control genomes. We detected only a single private homozygous protein-changing variant in the critical intervals. The detected variant was a 4 bp deletion, c.2717_2720delACAG, in the AKNA gene encoding the AT-hook transcription factor. It causes a frame-shift introducing a premature stop codon and truncates 37% of the open reading frame, p.(Asp906Alafs*173). We genotyped 88 Rough Collies consisting of family members and unrelated individuals. All three available cases were homozygous for the mutant allele and all 85 non-affected dogs were either homozygous wildtype (n = 67) or heterozygous (n = 18). AKNA modulates inflammatory immune responses. Akna−/− knockout mice die shortly after birth due to systemic autoimmune inflammatory processes including lung inflammation that is accompanied by enhanced leukocyte infiltration and alveolar destruction. The perfect genotype-phenotype association and the comparative functional data strongly suggest that the detected AKNA:c.2717_2720delACAG variant caused the observed severe airway inflammation in the investigated dogs. Our findings enable genetic testing, which can be used to avoid the unintentional breeding of affected puppies.

scholarly journals Loss of Function of the GdpP Protein Leads to Joint β-Lactam/Glycopeptide Tolerance in Staphylococcus aureus

2011 ◽  
Vol 56 (1) ◽  
pp. 579-581 ◽  
Author(s):  
J. M. Griffiths ◽  
A. J. O'Neill
Keyword(s):  
Signal Transduction ◽  
Staphylococcus Aureus ◽  
Genome Sequencing ◽  
Genetic Basis ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Content Type ◽  
Peptidoglycan Biosynthesis ◽  
Insertional Inactivation

ABSTRACTThe genetic basis of tolerance to inhibitors of peptidoglycan biosynthesis inStaphylococcus aureuswas investigated by generating tolerant mutantsin vitroand characterizing them by comparative genome sequencing. Two independently selected tolerant mutants harbored nonsynonymous mutations ingdpP, a gene encoding a putative membrane-located signaling protein. Insertional inactivation ofgdpPalso conferred tolerance. Our findings further implicate altered signal transduction as a route to antibiotic tolerance inS. aureus.

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