scholarly journals Comprehensive Identification of Mutations Responsible for Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA)-to-VISA Conversion in Laboratory-Generated VISA Strains Derived from hVISA Clinical Strain Mu3

2013 ◽  
Vol 57 (12) ◽  
pp. 5843-5853 ◽  
Author(s):  
Miki Matsuo ◽  
Longzhu Cui ◽  
Jeeyoung Kim ◽  
Keiichi Hiramatsu
Keyword(s):  
Staphylococcus Aureus ◽  
Vancomycin Resistance ◽  
Clinical Strain ◽  
Single Mutation ◽  
Content Type ◽  
Mutant Strains ◽  
Genome Study ◽  
One Step ◽  
Cytidylate Kinase ◽  
Sizable Number

ABSTRACTHeterogeneous vancomycin-intermediateStaphylococcus aureus(hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10−6or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes,rpoB,rpoC,walK,pbp4, andpp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) wascmk, which encodes cytidylate kinase, followed closely byrpoB(5 out of 32), encoding the β subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number ofcmkmutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity incmkmutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate.

scholarly journals Characterization of the New AmpC β-Lactamase FOX-8 Reveals a Single Mutation, Phe313Leu, Located in the R2 Loop That Affects Ceftazidime Hydrolysis

2013 ◽  
Vol 57 (10) ◽  
pp. 5158-5161 ◽  
Author(s):  
Francisco José Pérez-Llarena ◽  
Frédéric Kerff ◽  
Laura Zamorano ◽  
María Carmen Fernández ◽  
Maria Luz Nuñez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Analysis ◽  
Clinical Strain ◽  
Single Mutation ◽  
Content Type ◽  
E Coli ◽  
Fold Reduction ◽  
Class C

ABSTRACTA novel class C β-lactamase (FOX-8) was isolated from a clinical strain ofEscherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. IsogenicE. colistrains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction inkcat/Kmfor ceftazidime compared to FOX-3. In the FOX family of β-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype ofE. colistrains carrying this variant.

scholarly journals Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Orla M. Fleury ◽  
Maeve A. McAleer ◽  
Cécile Feuillie ◽  
Cécile Formosa-Dague ◽  
Emily Sansevere ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Atopic Dermatitis ◽  
Clonal Complex ◽  
Ex Vivo ◽  
Nasal Carriage ◽  
Binding Activity ◽  
Clinical Strain ◽  
Healthy Children ◽  
Content Type ◽  
Force Microscopy

ABSTRACT Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.

scholarly journals Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption

2015 ◽  
Vol 81 (13) ◽  
pp. 4295-4305 ◽  
Author(s):  
Thomas Denes ◽  
Henk C. den Bakker ◽  
Jeffrey I. Tokman ◽  
Claudia Guldimann ◽  
Martin Wiedmann
Keyword(s):  
Listeria Monocytogenes ◽  
Mutant Strain ◽  
Teichoic Acid ◽  
Resistant Mutant ◽  
Single Mutation ◽  
Spontaneous Mutant ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Mutant Strains ◽  
Phage Resistant Mutant

ABSTRACTListeria-infecting phages are readily isolated fromListeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface ofListeria monocytogenesstrain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n= 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds toN-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminalN-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possessN-acetylglucosamine in their WTA.

scholarly journals Genomic Analysis of the Emergence of Vancomycin-Resistant Staphylococcus aureus

mBio ◽  
2012 ◽  
Vol 3 (4) ◽  
Author(s):  
Scott D. Kobayashi ◽  
James M. Musser ◽  
Frank R. DeLeo
Keyword(s):  
United States ◽  
Staphylococcus Aureus ◽  
Dna Sequences ◽  
Clonal Complex ◽  
Infectious Agent ◽  
The United States ◽  
Vancomycin Resistance ◽  
Genetic Characteristics ◽  
Content Type ◽  
Vancomycin Resistant

ABSTRACT Staphylococcus aureus is a human commensal bacterium and a prominent cause of infections globally. The high incidence of S. aureus infections is compounded by the ability of the microbe to readily acquire resistance to antibiotics. In the United States, methicillin-resistant S. aureus (MRSA) is a leading cause of morbidity and mortality by a single infectious agent. Therapeutic options for severe MRSA infections are limited to a few antibiotics to which the organism is typically susceptible, including vancomycin. Acquisition of high-level vancomycin resistance by MRSA is a major concern, but to date, there have been only 12 vancomycin-resistant S. aureus (VRSA) isolates reported in the United States and all belong to a phylogenetic lineage known as clonal complex 5. To gain enhanced understanding of the genetic characteristics conducive to the acquisition of vancomycin resistance by S. aureus , V. N. Kos et al. performed whole-genome sequencing of all 12 VRSA isolates and compared the DNA sequences to the genomes of other S. aureus strains. The findings provide new information about the evolutionary history of VRSA and identify genetic features that may bear on the relationship between S. aureus clonal complex 5 strains and the acquisition of vancomycin resistance genes from enterococci.

scholarly journals “Slow VISA,” a Novel Phenotype of Vancomycin Resistance, FoundIn Vitroin Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Strain Mu3

2014 ◽  
Vol 58 (9) ◽  
pp. 5024-5035 ◽  
Author(s):  
Michie Saito ◽  
Yuki Katayama ◽  
Tomomi Hishinuma ◽  
Akira Iwamoto ◽  
Yoshifumi Aiba ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Aureus Strain ◽  
Clinical Strain ◽  
Regulatory Mutation ◽  
Content Type ◽  
Extremely High Frequency ◽  
Vancomycin Mics ◽  
Autolytic Activity ◽  
Doubling Times

ABSTRACTHeterogeneous vancomycin-intermediateStaphylococcus aureus(hVISA) clinical strain Mu3 spontaneously generates VISA strains at an extremely high frequency (≥1 × 10−6). The generated VISA strains usually grow more slowly than does the parent hVISA strain, but they form colonies on vancomycin-containing agar plates before 48 h of incubation. However, we noticed a curious group of VISA strains, designated “slow VISA” (sVISA), whose colonies appear only after 72 h of incubation. They have extremely prolonged doubling times but have vancomycin MICs of 8 to ∼24 mg/liter when determined after 72 to ∼144 h of incubation. We established strain Mu3-6R-P (6R-P), which has a vancomycin MIC of 16 mg/liter (at 72 h), as a representative sVISA strain. Its cell wall was thickened and autolytic activity was decreased compared to the respective qualities of the parent hVISA strain Mu3. Whole-genome sequencing of 6R-P revealed only one mutation, encoded byrpoB(R512P), which replaced the 512th arginine of the RNA polymerase β-subunit with proline. Its VISA phenotype was unstable, and the strain frequently reverted to hVISA with concomitant losses of pinpoint colony morphology and cell wall thickness and reduced autolytic activity. Sequencing of therpoBgenes of the phenotypic revertant strains revealed mutations affecting the 512th codon, where the proline of 6R-P was replaced with leucine, serine, or histidine. Slow VISA generated in the tissues of an infected patient serves as a temporary shelter for hVISA to survive vancomycin therapy. The sVISA strain spontaneously returns to hVISA when the threat of vancomycin is lifted. TherpoB(R512P) mutation may be regarded as a regulatory mutation that switches the reversible phenotype of sVISA on and off.

scholarly journals Complete Reconstitution of the Vancomycin-Intermediate Staphylococcus aureus Phenotype of Strain Mu50 in Vancomycin-Susceptible S. aureus

2016 ◽  
Vol 60 (6) ◽  
pp. 3730-3742 ◽  
Author(s):  
Yuki Katayama ◽  
Miwa Sekine ◽  
Tomomi Hishinuma ◽  
Yoshifumi Aiba ◽  
Keiichi Hiramatsu
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Mutant Strain ◽  
Vancomycin Resistance ◽  
Wall Thickening ◽  
Cell Physiology ◽  
Content Type ◽  
Sequence Deletion ◽  
Quadruple Mutant ◽  
Genetic Events

Complete reconstitution of the vancomycin-intermediateStaphylococcus aureus(VISA) phenotype of strain Mu50 was achieved by sequentially introducing mutations into six genes of vancomycin-susceptibleS. aureus(VSSA) strain N315ΔIP. The six mutated genes were detected in VISA strain Mu50 but not in N315ΔIP. Introduction of the mutation Ser329Leu intovraS, encoding the sensor histidine kinase of thevraSRtwo-component regulatory (TCR) system, and another mutation, Glu146Lys, intomsrR, belonging to the LytR-CpsA-Psr (LCP) family, increased the level of vancomycin resistance to that detected in heterogeneous vancomycin-intermediateS. aureus(hVISA) strain Mu3. Introduction of two more mutations, Asn197Ser intograRof thegraSRTCR system and His481Tyr intorpoB, encoding the β subunit of RNA polymerase, converted the hVISA strain into a VISA strain with the same level of vancomycin resistance as Mu50. Surprisingly, however, the constructed quadruple mutant strain ΔIP4 did not have a thickened cell wall, a cardinal feature of the VISA phenotype. Subsequent study showed that cell wall thickening was an inducible phenotype in the mutant strain, whereas it was a constitutive one in Mu50. Finally, introduction of the Ala297Val mutation intofdh2, which encodes a putative formate dehydrogenase, or a 67-amino-acid sequence deletion intosle1[sle1(Δ67aa)], encoding the hydrolase ofN-acetylmuramyl-l-alanine amidase in the peptidoglycan, converted inducible cell wall thickening into constitutive cell wall thickening.sle1(Δ67aa) was found to cause a drastic decrease in autolysis activity. Thus, all six mutated genes required for acquisition of the VISA phenotype were directly or indirectly involved in the regulation of cell physiology. The VISA phenotype seemed to be achieved through multiple genetic events accompanying drastic changes in cell physiology.

scholarly journals Exposure of Staphylococcus aureus to Subinhibitory Concentrations of β-Lactam Antibiotics Induces Heterogeneous Vancomycin-Intermediate Staphylococcus aureus

2014 ◽  
Vol 58 (9) ◽  
pp. 5306-5314 ◽  
Author(s):  
Mélanie Roch ◽  
Perrine Clair ◽  
Adriana Renzoni ◽  
Marie-Elisabeth Reverdy ◽  
Olivier Dauwalder ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Prolonged Exposure ◽  
Brain Heart Infusion ◽  
Vancomycin Resistance ◽  
Beta Lactam ◽  
Glycopeptide Resistance ◽  
Content Type ◽  
Beta Lactam Antibiotics ◽  
Subinhibitory Concentrations

ABSTRACTGlycopeptides are known to select for heterogeneous vancomycin-intermediateStaphylococcus aureus(h-VISA) from susceptible strains. In certain clinical situations, h-VISA strains have been isolated from patients without previous exposure to glycopeptides, such as cystic fibrosis patients, who frequently receive repeated treatments with beta-lactam antibiotics. Our objective was to determine whether prolonged exposure to beta-lactam antibiotics can induce h-VISA. We exposed 3 clinical vancomycin-susceptible methicillin-resistantStaphylococcus aureus(MRSA) strains to ceftazidime, ceftriaxone, imipenem, and vancomycin (as a control) at subinhibitory concentrations for 18 daysin vitro. Population analyses showed progressive increases in vancomycin resistance; seven of the 12 derived strains obtained after induction were classified as h-VISA according to the following criteria: area under the curve (AUC) on day 18/AUC of Mu3 of ≥90% and/or growth on brain heart infusion (BHI) agar with 4 mg/liter vancomycin. The derived isolates had thickened cell walls proportional to the level of glycopeptide resistance. Genes known to be associated with glycopeptide resistance (vraSR,yvqF, SA1703,graRS,walKR, andrpoB) were PCR sequenced; node novomutations were observed upon beta-lactam exposure. To determine whethertrfA, a gene encoding a glycopeptide resistance factor, was essential in the selection of h-VISA upon beta-lactam pressure, atrfA-knockout strain was generated by allelic replacement. Indeed, beta-lactam exposure of this mutated strain showed no capacity to induce vancomycin resistance. In conclusion, these results showed that beta-lactam antibiotics at subinhibitory concentrations can induce intermediate vancomycin resistancein vitro. This induction required an intacttrfAlocus. Our results suggest that prior use of beta-lactam antibiotics can compromise vancomycin efficacy in the treatment of MRSA infections.

scholarly journals VraR Binding to the Promoter Region of agr Inhibits Its Function in Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Yuanyuan Dai ◽  
Wenjiao Chang ◽  
Changcheng Zhao ◽  
Jing Peng ◽  
Liangfei Xu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Promoter Region ◽  
Reference Strain ◽  
Vancomycin Resistance ◽  
Mobility Shift ◽  
Content Type ◽  
Promoter Sequences ◽  
Dnase I Footprinting ◽  
Resistant Strains ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Acquisition of vancomycin resistance in Staphylococcus aureus is often accompanied by a reduction in virulence, but the mechanisms underlying this change remain unclear. The present study was undertaken to investigate this process in a clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain, 10827; an hVISA reference strain, Mu3; and a VISA reference strain, Mu50, along with their respective series of vancomycin-induced resistant strains. In these strains, increasing MICs of vancomycin were associated with increased expression of the vancomycin resistance-associated regulator gene (vraR) and decreased expression of virulence genes (hla, hlb, and coa) and virulence-regulated genes (RNAIII, agrA, and saeR). These results suggested that VraR might have a direct or indirect effect on virulence in S. aureus. In electrophoretic mobility shift assays, VraR did not bind to promoter sequences of hla, hlb, and coa genes, but it did bind to the agr promoter region. In DNase I footprinting assays, VraR protected a 15-nucleotide (nt) sequence in the intergenic region between the agr P2 and P3 promoters. These results indicated that when S. aureus is subject to induction by vancomycin, expression of vraR is upregulated, and VraR binding inhibits the function of the Agr quorum-sensing system, causing reductions in the virulence of VISA/hVISA strains. Our results suggested that VraR in S. aureus is involved not only in the regulation of vancomycin resistance but also in the regulation of virulence.

scholarly journals Structural Basis of Reduced Susceptibility to Ceftazidime-Avibactam and Cefiderocol in Enterobacter cloacae Due to AmpC R2 Loop Deletion

2020 ◽  
Vol 64 (7) ◽  
Author(s):  
Akito Kawai ◽  
Christi L. McElheny ◽  
Alina Iovleva ◽  
Ellen G. Kline ◽  
Nicolas Sluis-Cremer ◽  
...  
Keyword(s):  
Structural Changes ◽  
Enterobacter Cloacae ◽  
Multidrug Resistant ◽  
Structural Basis ◽  
Clinical Strain ◽  
Resistant Bacteria ◽  
Single Mutation ◽  
Close Monitoring ◽  
Content Type ◽  
Hydrolysis Of

ABSTRACT Ceftazidime-avibactam and cefiderocol are two of the latest generation β-lactam agents that possess expanded activity against highly drug-resistant bacteria, including carbapenem-resistant Enterobacterales. Here, we show that structural changes in AmpC β-lactamases can confer reduced susceptibility to both agents. A multidrug-resistant Enterobacter cloacae clinical strain (Ent385) was found to be resistant to ceftazidime-avibactam and cefiderocol without prior exposure to either agent. The AmpC β-lactamase of Ent385 (AmpCEnt385) contained an alanine-proline deletion at positions 294 and 295 (A294_P295del) in the R2 loop. AmpCEnt385 conferred reduced susceptibility to ceftazidime-avibactam and cefiderocol when cloned into Escherichia coli TOP10. Purified AmpCEnt385 showed increased hydrolysis of ceftazidime and cefiderocol compared to AmpCEnt385Rev, in which the deletion was reverted. Comparisons of crystal structures of AmpCEnt385 and AmpCP99, the canonical AmpC of E. cloacae complex, revealed that the two-residue deletion in AmpCEnt385 induced drastic structural changes of the H-9 and H-10 helices and the R2 loop, which accounted for the increased hydrolysis of ceftazidime and cefiderocol. The potential for a single mutation in ampC to confer reduced susceptibility to both ceftazidime-avibactam and cefiderocol requires close monitoring.

scholarly journals Complete Genome Sequence of a Community-Associated Methicillin-Resistant Staphylococcus aureus Hypervirulent Strain, USA300-C2406, Isolated from a Patient with a Lethal Case of Necrotizing Pneumonia

2017 ◽  
Vol 5 (22) ◽  
Author(s):  
Jo-Ann McClure ◽  
Kunyan Zhang
Keyword(s):  
Staphylococcus Aureus ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Clinical Strain ◽  
Necrotizing Pneumonia ◽  
Significant Morbidity ◽  
Methicillin Resistant ◽  
Content Type

ABSTRACT USA300 is a predominant community-associated methicillin-resistant Staphylococcus aureus strain causing significant morbidity and mortality. We present here the full annotated genome of a USA300 hypervirulent clinical strain, USA300-C2406, isolated from a patient with a lethal case of necrotizing pneumonia, to gain a better understanding of USA300 hypervirulence.

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