scholarly journals A Mutation of RNA Polymerase β′ Subunit (RpoC) Converts Heterogeneously Vancomycin-Intermediate Staphylococcus aureus (hVISA) into “Slow VISA”

2015 ◽  
Vol 59 (7) ◽  
pp. 4215-4225 ◽  
Author(s):  
Miki Matsuo ◽  
Tomomi Hishinuma ◽  
Yuki Katayama ◽  
Keiichi Hiramatsu
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Rna Polymerase ◽  
Stringent Response ◽  
Whole Genome ◽  
Multicopy Plasmid ◽  
Β Subunit ◽  
Whole Genome Analysis ◽  
Content Type ◽  
Peptidoglycan Biosynthesis

ABSTRACTVarious mutations in therpoBgene, which encodes the RNA polymerase β subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediateStaphylococcus aureus(VISA) and heterogeneously VISA (hVISA) strains. We reported thatrpoBmutations are also linked to the expression of the recently found “slow VISA” (sVISA) phenotype (M. Saito, Y. Katayama, T. Hishinuma, A. Iwamoto, Y. Aiba, K Kuwahara-Arai, L. Cui, M. Matsuo, N. Aritaka, and K. Hiramatsu, Antimicrob Agents Chemother 58:5024–5035, 2014,http://dx.doi.org/10.1128/AAC.02470-13). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of therpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (DT) (72 min) and high vancomycin MIC (16 mg/liter). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of low growth rate, cell wall thickness, and reduced autolysis. Whole-genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation, F562L, inrpoC. Introduction of the wild-type (WT)rpoCgene using a multicopy plasmid resolved the sVISA phenotype of V6-5, indicating that therpoC(P440L) mutant expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated an additional 10 revertants to hVISA and VISA. In subsequent whole-genome analysis, we identified compensatory mutations in the genes of three distinct functional categories: therpoCgene itself as regulatory mutations, peptidoglycan biosynthesis genes, andrelQ, which is involved in the stringent response. It appears that therpoC(P440L) mutation causes the sVISA phenotype by augmenting cell wall peptidoglycan synthesis and through the control of the stringent response.

scholarly journals Mutation of RNA Polymerase β-Subunit Gene Promotes Heterogeneous-to-Homogeneous Conversion of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus

2013 ◽  
Vol 57 (10) ◽  
pp. 4861-4871 ◽  
Author(s):  
Yoshifumi Aiba ◽  
Yuki Katayama ◽  
Tomomi Hishinuma ◽  
Hiroko Murakami-Kuroda ◽  
Longzhu Cui ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Rna Polymerase ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Phenotypic Expression ◽  
Amino Acid Replacement ◽  
Whole Genome ◽  
Β Subunit ◽  
Methicillin Resistant ◽  
Content Type

ABSTRACTThree types of phenotypic expression of β-lactam resistance have been reported in methicillin-resistantStaphylococcus aureus(MRSA): heterogeneous, homogeneous, and Eagle-type resistance. Heterogeneous-to-homogeneous conversion of β-lactam resistance is postulated to be caused by a chromosomal mutation (chr*) in addition to the expression of themecAgene. Eagle-type resistance is a unique phenotype ofchr*occurring in pre-MRSA strain N315 whosemecAgene expression is strongly repressed by an intactmecIgene. We here report that certain mutations of therpoBgene, encoding the RNA polymerase β subunit, belong tochr*. We studied homogeneous MRSA (homo-MRSA) strain N315ΔIP-H5 (abbreviated as ΔIP-H5), which was obtained from hetero-MRSA strain N315ΔIP by selection with 8 mg/liter imipenem. Whole-genome sequencing of ΔIP-H5 revealed the presence of a unique mutation in therpoBgene,rpoB(N967I), causing the amino acid replacement of Asn by Ile at position 967 of RpoB. The effect of therpoB(N967I) mutation was confirmed by constructing a revertant H5rpoB(I967N) strain as well as an N315-derived mutant, N315rpoB(N967I). H5rpoB(I967N) regained the hetero-resistance phenotype, and the N315rpoB(N967I) strain showed an Eagle-type phenotype similar to that of the typical Eagle-type MRSA strain N315h4. Furthermore, subsequent whole-genome sequencing revealed that N315h4 also had a missense mutation ofrpoB(R644H). Introduction of therpoB(N967I) mutation was accompanied by decreased autolysis, prolonged doubling time, and tolerance to bactericidal concentrations of methicillin. We consider thatrpoBmutations are the major cause for heterogeneous-to-homogeneous phenotypic conversion of β-lactam resistance in MRSA strain N315 and its derived strains.

scholarly journals Reconstruction ofmreBExpression in Staphylococcus aureus via a Collection of New Integrative Plasmids

2014 ◽  
Vol 80 (13) ◽  
pp. 3868-3878 ◽  
Author(s):  
Ana Yepes ◽  
Gudrun Koch ◽  
Andrea Waldvogel ◽  
Juan-Carlos Garcia-Betancur ◽  
Daniel Lopez
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Biology ◽  
Genetic Manipulation ◽  
Protein Localization ◽  
Antibiotic Resistance Genes ◽  
Wall Material ◽  
Content Type ◽  
Genetic Manipulations ◽  
Discrete Structures

ABSTRACTProtein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial modelsEscherichia coliandBacillus subtilishave been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacteriumStaphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of theS. aureuschromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression ofmreBinS. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that inS. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the useS. aureusas a model system in exploring diverse aspects of cellular microbiology.

scholarly journals Whole Genome Analysis of Epidemiologically Closely Related Staphylococcus aureus Isolates

PLoS ONE ◽  
2013 ◽  
Vol 8 (10) ◽  
pp. e78340 ◽  
Author(s):  
Maarten Schijffelen ◽  
Sergey R. Konstantinov ◽  
Gérard Lina ◽  
Iris Spiliopoulou ◽  
Engeline van Duijkeren ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Genome Analysis ◽  
Whole Genome ◽  
Whole Genome Analysis

scholarly journals Novel Combinations of Vancomycin plus Ceftaroline or Oxacillin against Methicillin-Resistant Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA

2013 ◽  
Vol 57 (5) ◽  
pp. 2376-2379 ◽  
Author(s):  
B. J. Werth ◽  
C. Vidaillac ◽  
K. P. Murray ◽  
K. L. Newton ◽  
G. Sakoulas ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Inverse Correlation ◽  
Fluorescent Dye ◽  
Beta Lactam ◽  
Significant Inverse Correlation ◽  
Methicillin Resistant ◽  
Content Type ◽  
Time Kill ◽  
Wall Interactions

ABSTRACTWe demonstrated a significant inverse correlation between vancomycin and beta-lactam susceptibilities in vancomycin-intermediateStaphylococcus aureus(VISA) and heterogeneous VISA (hVISA) isolates. Using time-kill assays, vancomycin plus oxacillin or ceftaroline was synergistic against 3 of 5 VISA and 1 of 5 hVISA isolates or 5 of 5 VISA and 4 of 5 hVISA isolates, respectively. Beta-lactam exposure reduced overall vancomycin-Bodipy (dipyrrometheneboron difluoride [4,4-difluoro-4-bora-3a,4a-diaza-s-indacene] fluorescent dye) binding but may have improved vancomycin-cell wall interactions to improve vancomycin activity. Further research is warranted to elucidate the mechanism behind vancomycin and beta-lactam synergy.

scholarly journals Biosynthesis of the Unique Wall Teichoic Acid of Staphylococcus aureus Lineage ST395

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Volker Winstel ◽  
Patricia Sanchez-Carballo ◽  
Otto Holst ◽  
Guoqing Xia ◽  
Andreas Peschel
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Biosynthesis Pathway ◽  
Glycerol Phosphate ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Wall Teichoic Acids

ABSTRACT The major clonal lineages of the human pathogen Staphylococcus aureus produce cell wall-anchored anionic poly-ribitol-phosphate (RboP) wall teichoic acids (WTA) substituted with d-Alanine and N-acetyl-d-glucosamine. The phylogenetically isolated S. aureus ST395 lineage has recently been found to produce a unique poly-glycerol-phosphate (GroP) WTA glycosylated with N-acetyl-d-galactosamine (GalNAc). ST395 clones bear putative WTA biosynthesis genes on a novel genetic element probably acquired from coagulase-negative staphylococci (CoNS). We elucidated the ST395 WTA biosynthesis pathway and identified three novel WTA biosynthetic genes, including those encoding an α-O-GalNAc transferase TagN, a nucleotide sugar epimerase TagV probably required for generation of the activated sugar donor substrate for TagN, and an unusually short GroP WTA polymerase TagF. By using a panel of mutants derived from ST395, the GalNAc residues carried by GroP WTA were found to be required for infection by the ST395-specific bacteriophage Φ187 and to play a crucial role in horizontal gene transfer of S. aureus pathogenicity islands (SaPIs). Notably, ectopic expression of ST395 WTA biosynthesis genes rendered normal S. aureus susceptible to Φ187 and enabled Φ187-mediated SaPI transfer from ST395 to regular S. aureus. We provide evidence that exchange of WTA genes and their combination in variable, mosaic-like gene clusters have shaped the evolution of staphylococci and their capacities to undergo horizontal gene transfer events. IMPORTANCE The structural highly diverse wall teichoic acids (WTA) are cell wall-anchored glycopolymers produced by most Gram-positive bacteria. While most of the dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate WTA, the recently described ST395 lineage produces a distinct poly-glycerol-phosphate WTA type resembling the WTA backbone of coagulase-negative staphylococci (CoNS). Here, we analyzed the ST395 WTA biosynthesis pathway and found new types of WTA biosynthesis genes along with an evolutionary link between ST395 and CoNS, from which the ST395 WTA genes probably originate. The elucidation of ST395 WTA biosynthesis will help to understand how Gram-positive bacteria produce highly variable WTA types and elucidate functional consequences of WTA variation.

scholarly journals Whole-Genome Sequencing Confirms that Burkholderia pseudomallei Multilocus Sequence Types Common to Both Cambodia and Australia Are Due to Homoplasy

2014 ◽  
Vol 53 (1) ◽  
pp. 323-326 ◽  
Author(s):  
Birgit De Smet ◽  
Derek S. Sarovich ◽  
Erin P. Price ◽  
Mark Mayo ◽  
Vanessa Theobald ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Analysis ◽  
Burkholderia Pseudomallei ◽  
Whole Genome ◽  
Whole Genome Analysis ◽  
Content Type ◽  
Sequence Types

Burkholderia pseudomalleiisolates with shared multilocus sequence types (STs) have not been isolated from different continents. We identified two STs shared between Australia and Cambodia. Whole-genome analysis revealed substantial diversity within STs, correctly identified the Asian or Australian origin, and confirmed that these shared STs were due to homoplasy.

scholarly journals Modified Oxacillin Resistant Staphylococcus aureus detected in neonatal intensive care patients

2021 ◽  
Author(s):  
Melissa R. Gitman ◽  
Bremy Alburquerque ◽  
Adriana van de Guchte ◽  
Mitchell J. Sullivan ◽  
Ajay Obla ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Intensive Care ◽  
Neonatal Intensive Care ◽  
Methicillin Resistance ◽  
Nonsynonymous Substitution ◽  
Whole Genome ◽  
Penicillin Binding Protein ◽  
Whole Genome Analysis ◽  
Intensive Care Patients ◽  
Clinical Laboratories

AbstractActive surveillance in our neonatal intensive care unit identified Staphylococcus aureus cultures from two infants with heterogeneity in methicillin resistance between isolated subclones lacking mecA and mecC. Whole-genome analysis of 4 modified (MODSA) and 4 methicillin-susceptible (MSSA) subclones for each culture identified either truncating mutations in the cyclic diadenosine monophosphate phosphodiesterase enzyme (GdpP), or a nonsynonymous substitution in penicillin binding protein 2 (PBP2). These cases highlight the difficulty in identifying non-mecA/non-mecC-mediated methicillin-resistance in clinical laboratories.

scholarly journals The Nature of Antibacterial Adaptive Immune Responses against Staphylococcus aureus Is Dependent on the Growth Phase and Extracellular Peptidoglycan

2019 ◽  
Vol 88 (1) ◽  
Author(s):  
Payal P. Balraadjsing ◽  
Lisbeth D. Lund ◽  
Yuri Souwer ◽  
Sebastian A. J. Zaat ◽  
Hanne Frøkiær ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
T Cell ◽  
Stationary Phase ◽  
Growth Phase ◽  
Cell Response ◽  
Th17 Cell ◽  
Exponential Phase ◽  
Th1 Cell ◽  
Content Type

ABSTRACT Staphylococcus aureus has evolved different strategies to evade the immune response, which play an important role in its pathogenesis. The bacteria express and shed various cell wall components and toxins during different stages of growth that may affect the protective T cell responses to extracellular and intracellular S. aureus. However, if and how the dendritic cell (DC)-mediated T cell response against S. aureus changes during growth of the bacterium remain elusive. In this study, we show that exponential-phase (EP) S. aureus bacteria were endocytosed very efficiently by human DCs, and these DCs strongly promoted production of the T cell polarizing factor interleukin-12 (IL-12). In contrast, stationary-phase (SP) S. aureus bacteria were endocytosed less efficiently by DCs, and these DCs produced small amounts of IL-12. The high level of IL-12 production induced by EP S. aureus led to the development of a T helper 1 (Th1) cell response, which was inhibited after neutralization of IL-12. Furthermore, preincubation with the staphylococcal cell wall component peptidoglycan (PGN), characteristically shed during the exponential growth phase, modulated the DC response to EP S. aureus. PGN preincubation appeared to inhibit IL-12p35 expression, leading to downregulation of IL-12 and an increase of IL-23 production by DCs, enhancing Th17 cell development. Taken together, our data indicate that exponential-phase S. aureus bacteria induce a stronger IL-12-dependent Th1 cell response than stationary-phase S. aureus and that this Th1 cell response shifted toward a Th17 cell response in the presence of PGN.

scholarly journals Whole-Genome Analysis of a Shiga Toxin-Producing Escherichia coli O103:H2 Strain Isolated from Cattle Feces

2020 ◽  
Vol 9 (45) ◽  
Author(s):  
Yujie Zhang ◽  
Yen-Te Liao ◽  
Vivian C. H. Wu
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Whole Genome Analysis ◽  
Content Type ◽  
Cattle Feces ◽  
Beef Products ◽  
Foodborne Outbreaks ◽  
Enterocyte Effacement

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) serotype O103 is one of the primary pathogenic contaminants of beef products, contributing to several foodborne outbreaks in recent years. Here, we report the whole-genome sequence of a STEC O103:H2 strain isolated from cattle feces that contains a locus of enterocyte effacement (LEE) pathogenicity island.

scholarly journals Identification and characterization of mutations responsible for the β-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tanit Boonsiri ◽  
Shinya Watanabe ◽  
Xin-Ee Tan ◽  
Kanate Thitiananpakorn ◽  
Ryu Narimatsu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quality Control ◽  
Protein Quality ◽  
Stringent Response ◽  
Protein Quality Control ◽  
Purine Biosynthesis ◽  
Genome Comparison ◽  
Whole Genome ◽  
Intergenic Regions ◽  
Mrsa Strains

Abstract Staphylococcus aureus strains that are susceptible to the β-lactam antibiotic oxacillin despite carrying mecA (OS-MRSA) cause serious clinical problems globally because of their ability to easily acquire β-lactam resistance. Understanding the genetic mechanism(s) of acquisition of the resistance is therefore crucial for infection control management. For this purpose, a whole-genome sequencing-based analysis was performed using 43 clinical OS-MRSA strains and 100 mutants with reduced susceptibility to oxacillin (MICs 1.0–256 µg/mL) generated from 26 representative OS-MRSA strains. Genome comparison between the mutants and their respective parent strains identified a total of 141 mutations in 46 genes and 8 intergenic regions. Among them, the mutations are frequently found in genes related to RNA polymerase (rpoBC), purine biosynthesis (guaA, prs, hprT), (p)ppGpp synthesis (relSau), glycolysis (pykA, fbaA, fruB), protein quality control (clpXP, ftsH), and tRNA synthase (lysS, gltX), whereas no mutations existed in mec and bla operons. Whole-genome transcriptional profile of the resistant mutants demonstrated that expression of genes associated with purine biosynthesis, protein quality control, and tRNA synthesis were significantly inhibited similar to the massive transcription downregulation seen in S. aureus during the stringent response, while the levels of mecA expression and PBP2a production were varied. We conclude that a combination effect of mecA upregulation and stringent-like response may play an important role in acquisition of β-lactam resistance in OS-MRSA.

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