scholarly journals Prolonged Growth of a Clinical Staphylococcus aureus Strain Selects for a Stable Small-Colony-Variant Cell Type

2014 ◽  
Vol 83 (2) ◽  
pp. 470-481 ◽  
Author(s):  
Long M. G. Bui ◽  
Peter Hoffmann ◽  
John D. Turnidge ◽  
Peter S. Zilm ◽  
Stephen P. Kidd
Keyword(s):  
Staphylococcus Aureus ◽  
Extracellular Matrix ◽  
Extracellular Dna ◽  
Aureus Strain ◽  
Specific Cell ◽  
Cell Type ◽  
Term Survival ◽  
Content Type ◽  
Prolonged Time ◽  
Small Colony

An undetermined feature ofStaphylococcus aureuspathogenesis is its persistence and then relapse of disease. This has been explained by its switch to alternative lifestyles, mainly as biofilm or small-colony variants (SCVs). Studying the native characteristics of SCVs has been problematic due to their reversion to the parental lifestyle. We have observed that for a number ofS. aureusstrains as they switch to an SCV lifestyle, there is the formation of an extracellular matrix. We focused our analysis on one strain, WCH-SK2. For bacterial survival in the host, the combination of low nutrients and the prolonged time frame forms a stress that selects for a specific cell type from the population. In this context, we used steady-state growth conditions with low nutrients and a controlled low growth rate for a prolonged time and with methylglyoxal. These conditions inducedS. aureusWCH-SK2 into a stable SCV cell type; the cells did not revert after subculturing. Analysis revealed these cells possessed a metabolic and surface profile that was different from those of previously described SCVs or biofilm cells. The extracellular matrix was protein and extracellular DNA but not polysaccharide. The SCV cells induced expression of certain surface proteins (such as Ebh) and synthesis of lantibiotics while downregulating factors that stimulate the immune response (leucocidin, capsule, and carotenoid). Our data reveal cell heterogeneity within anS. aureuspopulation and under conditions that resemble long-term survival in the host have identified a previously unnoticedS. aureuscell type with a distinctive metabolic and molecular profile.

scholarly journals Novel Insights into Staphylococcus aureus Deep Bone Infections: the Involvement of Osteocytes

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Dongqing Yang ◽  
Asiri R. Wijenayaka ◽  
Lucian B. Solomon ◽  
Stephen M. Pederson ◽  
David M. Findlay ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Infection Model ◽  
Cell Type ◽  
Content Type ◽  
Intracellular Infection ◽  
Joint Replacement Surgery ◽  
Modes Of Failure ◽  
Common Pathogen ◽  
Small Colony

ABSTRACT Periprosthetic joint infection (PJI) is a potentially devastating complication of orthopedic joint replacement surgery. PJI with associated osteomyelitis is particularly problematic and difficult to cure. Whether viable osteocytes, the predominant cell type in mineralized bone tissue, have a role in these infections is not clear, although their involvement might contribute to the difficulty in detecting and clearing PJI. Here, using Staphylococcus aureus , the most common pathogen in PJI, we demonstrate intracellular infection of human-osteocyte-like cells in vitro and S. aureus adaptation by forming quasi-dormant small-colony variants (SCVs). Consistent patterns of host gene expression were observed between in vitro -infected osteocyte-like cultures, an ex vivo human bone infection model, and bone samples obtained from PJI patients. Finally, we confirm S. aureus infection of osteocytes in clinical cases of PJI. Our findings are consistent with osteocyte infection being a feature of human PJI and suggest that this cell type may provide a reservoir for silent or persistent infection. We suggest that elucidating the molecular/cellular mechanism(s) of osteocyte-bacterium interactions will contribute to better understanding of PJI and osteomyelitis, improved pathogen detection, and treatment. IMPORTANCE Periprosthetic joint infections (PJIs) are increasing and are recognized as one of the most common modes of failure of joint replacements. Osteomyelitis arising from PJI is challenging to treat and difficult to cure and increases patient mortality 5-fold. Staphylococcus aureus is the most common pathogen causing PJI. PJI can have subtle symptoms and lie dormant or go undiagnosed for many years, suggesting persistent bacterial infection. Osteocytes, the major bone cell type, reside in bony caves and tunnels, the lacuno-canalicular system. We report here that S. aureus can infect and reside in human osteocytes without causing cell death both experimentally and in bone samples from patients with PJI. We demonstrate that osteocytes respond to infection by the differential regulation of a large number of genes. S. aureus adapts during intracellular infection of osteocytes by adopting the quasi-dormant small-colony variant (SCV) lifestyle, which might contribute to persistent or silent infection. Our findings shed new light on the etiology of PJI and osteomyelitis in general.

scholarly journals Genome Sequence of a Staphylococcus aureus Strain Isolated from a Dairy Cow That Was Nonresponsive to Antibiotic Treatment

2020 ◽  
Vol 9 (20) ◽  
Author(s):  
John D. Lippolis ◽  
Ellie J. Putz ◽  
Hao Ma ◽  
David P. Alt ◽  
Eduardo Casas ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dairy Cattle ◽  
Antibiotic Treatment ◽  
Genome Sequence ◽  
Dairy Cow ◽  
Aureus Strain ◽  
Chronic Case ◽  
Content Type

Staphylococcus aureus can cause mastitis in dairy cattle. We report the genome sequence of a Staphylococcus aureus strain isolated from a dairy cow with a chronic case of mastitis. The infection with this strain of Staphylococcus aureus was not cleared from the animal with antibiotic treatment.

scholarly journals A Two-Component System That Modulates Cyclic di-GMP Metabolism PromotesLegionella pneumophilaDifferentiation and Viability in Low-Nutrient Conditions

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Elisa D. Hughes ◽  
Brenda G. Byrne ◽  
Michele S. Swanson
Keyword(s):  
Life Cycle ◽  
Lifestyle Changes ◽  
Negative Regulator ◽  
Broth Culture ◽  
Cell Type ◽  
Two Component System ◽  
Term Survival ◽  
Component System ◽  
Content Type ◽  
Two Component

ABSTRACTDuring its life cycle, the environmental pathogenLegionella pneumophilaalternates between a replicative and transmissive cell type when cultured in broth, macrophages, or amoebae. Within a protozoan host,L. pneumophilafurther differentiates into the hardy cell type known as the mature infectious form (MIF). The second messenger cyclic di-GMP coordinates lifestyle changes in many bacterial species, but its role in theL. pneumophilalife cycle is less understood. Using anin vitrobroth culture model that approximates the intracellular transition from the replicative to the transmissive form, here we investigate the contribution toL. pneumophiladifferentiation of a two-component system (TCS) that regulates cyclic di-GMP metabolism. The TCS is encoded bylpg0278-lpg0277and is cotranscribed withlpg0279, which encodes a protein upregulated in MIF cells. The promoter for this operon is RpoS dependent and induced in nutrient-limiting conditions that do not support replication, as demonstrated using agfpreporter and quantitative PCR (qPCR). The response regulator of the TCS (Lpg0277) is a bifunctional enzyme that both synthesizes and degrades cyclic di-GMP. Using a panel of site-directed point mutants, we show that cyclic di-GMP synthesis mediated by a conserved GGDEF domain promotes growth arrest of replicativeL. pneumophila, accumulation of pigment and poly-3-hydroxybutyrate storage granules, and viability in nutrient-limiting conditions. Genetic epistasis tests predict that the MIF protein Lpg0279 acts as a negative regulator of the TCS. Thus,L. pneumophilais equipped with a regulatory network in which cyclic di-GMP stimulates the switch from a replicative to a resilient state equipped to survive in low-nutrient environments.IMPORTANCEAlthough an intracellular pathogen,L. pneumophilahas developed mechanisms to ensure long-term survival in low-nutrient aqueous conditions. Eradication ofL. pneumophilafrom contaminated water supplies has proven challenging, as outbreaks have been traced to previously remediated systems. Understanding the genetic determinants that supportL. pneumophilapersistence in low-nutrient environments can inform design and assessment of remediation strategies. Here we characterize a genetic locus that encodes a two-component signaling system (lpg0278-lpg0277) and a putative regulator protein (lpg0279) that modulates the production of the messenger molecule cyclic di-GMP. We show that this locus promotes bothL. pneumophilacell differentiation and survival in nutrient-limiting conditions, thus advancing the understanding of the mechanisms that contribute toL. pneumophilaenvironmental resilience.

scholarly journals CidR and CcpA Synergistically Regulate Staphylococcus aureus cidABC Expression

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Marat R. Sadykov ◽  
Ian H. Windham ◽  
Todd J. Widhelm ◽  
Vijaya Kumar Yajjala ◽  
Sean M. Watson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Carbon Metabolism ◽  
Protein A ◽  
Transcriptional Regulators ◽  
Extracellular Dna ◽  
Regulatory Control ◽  
Pcr Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Biofilm Development

ABSTRACT The death and lysis of a subpopulation of Staphylococcus aureus cells during biofilm development benefit the whole bacterial population through the release of an important component of the biofilm matrix, extracellular DNA. Previously, we have demonstrated that these processes are affected by the gene products of the cidABC operon, the expression of which is controlled by the LysR-type transcriptional regulator, CidR. In this study, we characterized cis- and trans-acting elements essential for the induction of the cidABC operon. In addition to a CidR-binding site located within the cidABC promoter region, sequence analysis revealed the presence of a putative catabolite responsive element (cre box), suggestive of the involvement of the catabolite control protein A (CcpA) in the regulation of cidABC expression. This was confirmed using electrophoretic mobility shift assays and real-time reverse transcriptase PCR analysis demonstrating the direct positive control of cidABC transcription by the master regulator of carbon metabolism. Furthermore, the importance of CcpA and the identified cre site for the induction of the cidABC operon was demonstrated by examining the expression of PcidABC-lacZ reporter fusions in various mutant strains in which the genes involved in carbon metabolism and carbon catabolite repression were disrupted. Together the results of this study demonstrate the necessity of both transcriptional regulators, CidR and CcpA, for the induction of the cidABC operon and reveal the complexity of molecular interactions controlling its expression. IMPORTANCE This work focuses on the characterization of cis- and trans-acting elements essential for the induction of the cidABC operon in S. aureus. The results of this study are the first to demonstrate the synergistic control of cidABC expression by transcriptional regulators CidR and CcpA during carbohydrate metabolism. We established that the full induction of cidABC expression depends on the metabolic state of bacteria and requires both CidR and CcpA. Together, these findings delineate regulatory control of cidABC expression under different metabolic conditions and provide important new insights into our understanding of cell death mechanisms during biofilm development in S. aureus.

scholarly journals Complete Genome Sequence of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Strain WCUH29

2019 ◽  
Vol 8 (23) ◽  
Author(s):  
Ting Lei ◽  
Ying Zhang ◽  
Junshu Yang ◽  
Kevin Silverstein ◽  
Yinduo Ji
Keyword(s):  
Staphylococcus Aureus ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Model System ◽  
Aureus Strain ◽  
Methicillin Resistant ◽  
Content Type ◽  
Hospital Acquired

The hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) strain WCUH29 has been intensively and widely used as a model system for identification and evaluation of novel antibacterial targets and pathogenicity. In this announcement, we report the complete genome sequence of HA-MRSA WCUH29 (NCIMB 40771).

scholarly journals Pushing beyond the Envelope: the Potential Roles of OprF inPseudomonas aeruginosaBiofilm Formation and Pathogenicity

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Erin K. Cassin ◽  
Boo Shan Tseng
Keyword(s):  
Extracellular Matrix ◽  
Outer Membrane ◽  
Biofilm Formation ◽  
Matrix Protein ◽  
Cell Envelope ◽  
Outer Membrane Vesicles ◽  
Extracellular Dna ◽  
Future Research ◽  
Content Type ◽  
Biofilm Matrix

ABSTRACTThe ability ofPseudomonas aeruginosato form biofilms, which are communities of cells encased in a self-produced extracellular matrix, protects the cells from antibiotics and the host immune response. While some biofilm matrix components, such as exopolysaccharides and extracellular DNA, are relatively well characterized, the extracellular matrix proteins remain understudied. Multiple proteomic analyses of theP. aeruginosasoluble biofilm matrix and outer membrane vesicles, which are a component of the matrix, have identified OprF as an abundant matrix protein. To date, the few reports on the effects ofoprFmutations on biofilm formation are conflicting, and little is known about the potential role of OprF in the biofilm matrix. The majority of OprF studies focus on the protein as a cell-associated porin. As a component of the outer membrane, OprF assumes dual conformations and is involved in solute transport, as well as cell envelope integrity. Here, we review the current literature on OprF inP. aeruginosa, discussing how the structure and function of the cell-associated and matrix-associated protein may affect biofilm formation and pathogenesis in order to inform future research on this understudied matrix protein.

scholarly journals Modulation of Staphylococcus aureus Biofilm Matrix by Subinhibitory Concentrations of Clindamycin

2016 ◽  
Vol 60 (10) ◽  
pp. 5957-5967 ◽  
Author(s):  
Katrin Schilcher ◽  
Federica Andreoni ◽  
Vanina Dengler Haunreiter ◽  
Kati Seidl ◽  
Barbara Hasse ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Treatment ◽  
Molecular Mechanisms ◽  
Treatment Strategies ◽  
Extracellular Dna ◽  
Content Type ◽  
Transcriptional Stress ◽  
Sigma Factor B ◽  
Subinhibitory Concentrations ◽  
Biofilm Matrix

ABSTRACTStaphylococcus aureusbiofilms are extremely difficult to treat. They provide a protected niche for the bacteria, rendering them highly recalcitrant toward host defenses as well as antibiotic treatment. Bacteria within a biofilm are shielded from the immune system by the formation of an extracellular polymeric matrix, composed of polysaccharides, extracellular DNA (eDNA), and proteins. Many antibiotics do not readily penetrate biofilms, resulting in the presence of subinhibitory concentrations of antibiotics. Here, we show that subinhibitory concentrations of clindamycin triggered a transcriptional stress response inS. aureusvia the alternative sigma factor B (σB) and upregulated the expression of the major biofilm-associated genesatlA,lrgA,agrA, thepsmgenes,fnbA, andfnbB. Our data suggest that subinhibitory concentrations of clindamycin alter the ability ofS. aureusto form biofilms and shift the composition of the biofilm matrix toward higher eDNA content. An understanding of the molecular mechanisms underlying biofilm assembly and dispersal in response to subinhibitory concentrations of clinically relevant antibiotics such as clindamycin is critical to further optimize antibiotic treatment strategies of biofilm-associatedS. aureusinfections.

scholarly journals Inactivation of thyA in Staphylococcus aureus Attenuates Virulence and Has a Strong Impact on Metabolism and Virulence Gene Expression

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Andre Kriegeskorte ◽  
Desiree Block ◽  
Mike Drescher ◽  
Nadine Windmüller ◽  
Alexander Mellmann ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Thymidylate Synthase ◽  
Molecular Basis ◽  
De Novo ◽  
Strong Impact ◽  
Small Colony Variants ◽  
Content Type ◽  
Long Term Treatment ◽  
Small Colony ◽  
The Impact

ABSTRACTStaphylococcus aureusthymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronicS. aureusinfections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS;thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations inthyAwere leading to inactivity of TS proteins, and TS inactivity led to tremendous impact onS. aureusphysiology and virulence. Whole DNA microarray analysis of the constructed ΔthyAmutant identified severe alterations compared to the wild type. Important virulence regulators (agr,arlRS,sarA) and major virulence determinants (hla,hlb,sspAB, andgeh) were downregulated, while genes important for colonization (fnbA,fnbB,spa,clfB,sdrC, andsdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyAmutant was strongly attenuated in virulence models, including aCaenorhabditis eleganskilling model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed thatthyAactivity has a major role forS. aureusvirulence and physiology.IMPORTANCEThymidine-dependent small-colony variants (TD-SCVs) ofStaphylococcus aureuscarry mutations in the thymidylate synthase (TS) gene (thyA) responsible forde novosynthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistantS. aureus, the therapeutic use of TMP-SMX is increasing. Today, the emergence of TD-SCVs is still underestimated due to misidentification in the diagnostic laboratory. This study showed for the first time that mutational inactivation of TS is the molecular basis for the TD-SCV phenotype and that TS inactivation has a strong impact onS. aureusvirulence and physiology. Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

scholarly journals OptimizedIn VitroAntibiotic Susceptibility Testing Method for Small-Colony Variant Staphylococcus aureus

2016 ◽  
Vol 60 (3) ◽  
pp. 1725-1735 ◽  
Author(s):  
Mimi R. Precit ◽  
Daniel J. Wolter ◽  
Adam Griffith ◽  
Julia Emerson ◽  
Jane L. Burns ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Susceptibility Testing ◽  
Brain Heart Infusion ◽  
Chronic Infections ◽  
Poor Growth ◽  
Content Type ◽  
Treatment History ◽  
Mueller Hinton ◽  
Mueller Hinton Agar ◽  
Small Colony

Staphylococcus aureussmall-colony variants (SCVs) emerge frequently during chronic infections and are often associated with worse disease outcomes. There are no standardized methods for SCV antibiotic susceptibility testing (AST) due to poor growth and reversion to normal-colony (NC) phenotypes on standard media. We sought to identify reproducible methods for AST ofS. aureusSCVs and to determine whether SCV susceptibilities can be predicted on the basis of treatment history, SCV biochemical type (auxotrophy), or the susceptibilities of isogenic NC coisolates. We tested the growth and stability of SCV isolates on 11 agar media, selecting for AST 2 media that yielded optimal SCV growth and the lowest rates of reversion to NC phenotypes. We then performed disk diffusion AST on 86S. aureusSCVs and 28 isogenic NCs and Etest for a subset of 26 SCVs and 24 isogenic NCs. Growth and reversion were optimal on brain heart infusion agar and Mueller-Hinton agar supplemented with compounds for which most clinical SCVs are auxotrophic: hemin, menadione, and thymidine. SCVs were typically nonsusceptible to either trimethoprim-sulfamethoxazole or aminoglycosides, in accordance with the auxotrophy type. In contrast, SCVs were variably nonsusceptible to fluoroquinolones, macrolides, lincosamides, fusidic acid, and rifampin;mecA-positive SCVs were invariably resistant to cefoxitin. All isolates (both SCVs and NCs) were susceptible to quinupristin-dalfopristin, vancomycin, minocycline, linezolid, chloramphenicol, and tigecycline. Analysis of SCV auxotrophy type, isogenic NC antibiograms, and antibiotic treatment history had limited utility in predicting SCV susceptibilities. With clinical correlation, this AST method and these results may prove useful in directing treatment for SCV infections.

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