scholarly journals NorB, an Efflux Pump in Staphylococcus aureus Strain MW2, Contributes to Bacterial Fitness in Abscesses

2008 ◽  
Vol 190 (21) ◽  
pp. 7123-7129 ◽  
Author(s):  
Yanpeng Ding ◽  
Yoshikuni Onodera ◽  
Jean C. Lee ◽  
David C. Hooper
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Single Copy ◽  
Aureus Strain ◽  
Bacterial Survival ◽  
Bacterial Fitness

ABSTRACT While remaining a major problem in hospitals, Staphylococcus aureus is now spreading in communities. Strain MW2 (USA400 lineage) and other community methicillin-resistant S. aureus strains most commonly cause skin infections with abscess formation. Multidrug resistance (MDR) efflux pumps contribute to antimicrobial resistance but may also contribute to bacterial survival by removal of environmental toxins. In S. aureus, NorA, NorB, NorC, and Tet38 are chromosomally encoded efflux pumps whose overexpression can confer MDR to quinolones and other compounds (Nor pumps) or tetracyclines alone (Tet38), but the natural substrates of these pumps are not known. To determine the role of these efflux pumps in a natural environment in the absence of antibiotics, we used strain MW2 in a mouse subcutaneous abscess model and compared pump gene expression as determined by reverse transcription-PCR in the abscesses and in vitro. norB and tet38 were selectively upregulated in vivo more than 171- and 24-fold, respectively, whereas norA and norC were downregulated. These changes were associated with an increase in expression of mgrA, which encodes a transcriptional regulator known to affect pump gene expression. In competition experiments using equal inocula of a norB or tet38 mutant and parent strain MW2, each mutant exhibited growth defects of about two- to threefold in vivo. In complementation experiments, a single-copy insertion of norB (but not a single-copy insertion of tet38) in the attB site within geh restored the growth fitness of the norB mutant in vivo. Our findings indicate that some MDR pumps, like NorB, can facilitate bacterial survival when they are overexpressed in a staphylococcal abscess and may contribute to the relative resistance of abscesses to antimicrobial therapy, thus linking bacterial fitness and resistance in vivo.

Expression of ERG11 and efflux pump genes CDR1, CDR2 and SNQ2 in voriconazole susceptible and resistant Candida glabrata strains

2019 ◽  
Vol 58 (1) ◽  
pp. 30-38
Author(s):  
Patricia Navarro-Rodríguez ◽  
Adela Martin-Vicente ◽  
Loida López-Fernández ◽  
Josep Guarro ◽  
Javier Capilla
Keyword(s):  
Gene Expression ◽  
Candida Glabrata ◽  
Efflux Pump ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Clear Correlation ◽  
Synonymous Mutations ◽  
First Time

AbstractCandida glabrata causes difficult to treat invasive candidiasis due to its antifungal resistance, mainly to azoles. The aim of the present work was to study the role of the genes ERG11, CDR1, CDR2, and SNQ2 on the resistance to voriconazole (VRC) in a set of C. glabrata strains with known in vitro and in vivo susceptibility to this drug. Eighteen clinical isolates of C. glabrata were exposed in vitro to VRC, and the expression of the cited genes was quantified by real time quantitative polymerase chain reaction (q-PCR). In addition, the ERG11 gene was amplified and sequenced to detect possible mutations. Ten synonymous mutations were found in 15 strains, two of them being reported for the first time; however, no amino acid changes were detected. ERG11 and CDR1 were the most expressed genes in all the strains tested, while the expression of CDR2 and SNQ2 was modest. Our results show that gene expression does not directly correlate with the VRC MIC. In addition, the expression profiles of ERG11 and efflux pump genes did not change consistently after exposure to VRC. Although individual analysis did not result in a clear correlation between MIC and gene expression, we did observe an increase in ERG11 and CDR1 expression in resistant strains. It is of interest that considering both in vitro and in vivo results, the slight increase in such gene expression correlates with the observed resistance to VRC.

scholarly journals 18β-Glycyrrhetinic Acid Inhibits Methicillin-Resistant Staphylococcus aureus Survival and Attenuates Virulence Gene Expression

2012 ◽  
Vol 57 (1) ◽  
pp. 241-247 ◽  
Author(s):  
Danyelle R. Long ◽  
Julia Mead ◽  
Jay M. Hendricks ◽  
Michele E. Hardy ◽  
Jovanka M. Voyich
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Virulence Gene ◽  
Glycyrrhetinic Acid ◽  
Licorice Root ◽  
Methicillin Resistant ◽  
Content Type ◽  
Virulence Gene Expression

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) has become a major source of infection in hospitals and in the community. Increasing antibiotic resistance inS. aureusstrains has created a need for alternative therapies to treat disease. A component of the licorice rootGlycyrrhizaspp., 18β-glycyrrhetinic acid (GRA), has been shown to have antiviral, antitumor, and antibacterial activity. This investigation explores thein vitroandin vivoeffects of GRA on MRSA pulsed-field gel electrophoresis (PFGE) type USA300. GRA exhibited bactericidal activity at concentrations exceeding 0.223 μM. Upon exposure ofS. aureusto sublytic concentrations of GRA, we observed a reduction in expression of key virulence genes, includingsaeRandhla. In murine models of skin and soft tissue infection, topical GRA treatment significantly reduced skin lesion size and decreased the expression ofsaeRandhlagenes. Our investigation demonstrates that at high concentrations GRA is bactericidal to MRSA and at sublethal doses it reduces virulence gene expression inS. aureusbothin vitroandin vivo.

scholarly journals Involvement of the AcrAB-TolC Efflux Pump in the Resistance, Fitness, and Virulence of Enterobacter cloacae

2012 ◽  
Vol 56 (4) ◽  
pp. 2084-2090 ◽  
Author(s):  
Astrid Pérez ◽  
Margarita Poza ◽  
Ana Fernández ◽  
Maria del Carmen Fernández ◽  
Susana Mallo ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Pathogenic Bacteria ◽  
Efflux Pump ◽  
Enterobacter Cloacae ◽  
Efflux Pumps ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type

ABSTRACTMultidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence inEnterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates ofE. cloacaewere used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). TheacrAandtolCgenes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrAand EcΔtolCand JcΔacrAand JcΔtolCknockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance ofE. cloacaeto several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that theacrAandtolCgenes both affect the fitness ofE. cloacae, as fitness was clearly reduced in theacrAandtolCKO strains. The median CI values obtainedin vitroandin vivowere, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in bothE. cloacaeclinical strains when either theacrAortolCgene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates ofE. cloacae.

scholarly journals Impacts of sarA and agr in Staphylococcus aureus Strain Newman on Fibronectin-Binding Protein A Gene Expression and Fibronectin Adherence Capacity In Vitro and in Experimental Infective Endocarditis

2004 ◽  
Vol 72 (3) ◽  
pp. 1832-1836 ◽  
Author(s):  
Yan-Qiong Xiong ◽  
Arnold S. Bayer ◽  
Michael R. Yeaman ◽  
Willem van Wamel ◽  
Adhar C. Manna ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Capacity ◽  
Protein A ◽  
Aureus Strain ◽  
Global Regulators ◽  
Fibronectin Binding Protein ◽  
Regulatory Loci ◽  
Fibronectin Binding

ABSTRACT We investigated the impacts of sarA and agr on fnbA expression and fibronectin-binding capacity in Staphylococcus aureus in vitro and in experimental endocarditis. Although sarA up-regulated and agr down-regulated both fnbA expression and fibronectin binding in vitro and in vivo, fnbA expression was positively regulated in the absence of both global regulators. Thus, additional regulatory loci contribute to fnbA regulation and fibronectin-binding capacities in S. aureus.

scholarly journals Simulated Antibiotic Exposures in anIn VitroHollow-Fiber Infection Model Influence Toxin Gene Expression and Production in Community-Associated Methicillin-Resistant Staphylococcus aureus Strain MW2

2011 ◽  
Vol 56 (1) ◽  
pp. 140-147 ◽  
Author(s):  
Solen Pichereau ◽  
Madhulatha Pantrangi ◽  
William Couet ◽  
Cedric Badiou ◽  
Gerard Lina ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Hollow Fiber ◽  
Aureus Strain ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infection Model ◽  
Toxin Gene ◽  
Polymorphonuclear Cells ◽  
Methicillin Resistant

ABSTRACTCommunity-associated methicillin-resistantStaphylococcus aureus(CA-MRSA) strain MW2 harbors a plethora of toxins to mediate its virulence. However, toxin expression and regulation with simulated clinical antimicrobial exposures are unclear. This study evaluated these relationships using anin vitropharmacodynamic hollow-fiber infection model. Clinical doses of clindamycin, linezolid, minocycline, trimethoprim-sulfamethoxazole (SXT), and vancomycin were simulated over 72 h against MW2 in the hollow fiber model. Expression levels oflukSF-PVand enterotoxin genessec4,sek,seq, andsel2were quantified by real-time PCR. Panton-Valentine leukocidin (PVL) was quantified by enzyme-linked immunosorbent assay (ELISA), and cytotoxicity was determined on polymorphonuclear cells (PMNs). Vancomycin produced the maximum MW2 killing (2.53 log10CFU/ml) after the first dose, but the greatest sustained killing over 72 h occurred with linezolid and clindamycin. Vancomycin and minocycline induced gene upregulation from 0 to 8 h, followed by downregulation for the remaining simulation period. Clindamycin decreased gene expression in the first 24 h, followed by moderate increases (2.5-fold) thereafter. Linezolid increased gene expression 11.4- to 200.4-fold but inhibited PVL production (0.6 ± 0.3 versus 5.9 ± 0.2 μg/ml, linezolid versus control at 72 h;P< 0.05). Similar effects on PVL production occurred with clindamycin and minocycline. SXT increased PVL production at 48 h (2.8-fold) and 72 h (4.9-fold) of treatment (P< 0.05), resulting in increased PVL cytotoxicity on PMNs. Linezolid, clindamycin, and minocycline were the most effective agents on decreasing the virulence potential in CA-MRSA, notably after 8 h of treatment. SXT had minimal effects on toxin gene regulation, but it increased production and cytotoxicity of PVL toxin in the model and may enhance virulence when it is used to treat severe infections.

scholarly journals Commensal bacteria augment Staphylococcus aureus infection by inactivation of phagocyte-derived reactive oxygen species

2021 ◽  
Vol 17 (9) ◽  
pp. e1009880
Author(s):  
Josie F. Gibson ◽  
Grace R. Pidwill ◽  
Oliver T. Carnell ◽  
Bas G. J. Surewaard ◽  
Daria Shamarina ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Staphylococcus Aureus ◽  
Reactive Oxygen ◽  
Cellular Level ◽  
Oxygen Species ◽  
Bacterial Survival ◽  
Efficacious Vaccine ◽  
Opportunist Pathogen

Staphylococcus aureus is a human commensal organism and opportunist pathogen, causing potentially fatal disease. The presence of non-pathogenic microflora or their components, at the point of infection, dramatically increases S. aureus pathogenicity, a process termed augmentation. Augmentation is associated with macrophage interaction but by a hitherto unknown mechanism. Here, we demonstrate a breadth of cross-kingdom microorganisms can augment S. aureus disease and that pathogenesis of Enterococcus faecalis can also be augmented. Co-administration of augmenting material also forms an efficacious vaccine model for S. aureus. In vitro, augmenting material protects S. aureus directly from reactive oxygen species (ROS), which correlates with in vivo studies where augmentation restores full virulence to the ROS-susceptible, attenuated mutant katA ahpC. At the cellular level, augmentation increases bacterial survival within macrophages via amelioration of ROS, leading to proliferation and escape. We have defined the molecular basis for augmentation that represents an important aspect of the initiation of infection.

scholarly journals Regulated Antisense RNA Eliminates Alpha-Toxin Virulence in Staphylococcus aureus Infection

1999 ◽  
Vol 181 (21) ◽  
pp. 6585-6590 ◽  
Author(s):  
Yinduo Ji ◽  
Andrea Marra ◽  
Martin Rosenberg ◽  
Gary Woodnutt
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Antisense Rna ◽  
Expression System ◽  
Toxin Gene ◽  
Critical Element ◽  
Bacterial Cells ◽  
Alpha Toxin

ABSTRACT The ability to selectively disrupt gene function remains a critical element in elucidating information regarding gene essentiality for bacterial growth and/or pathogenesis. In this study, we adapted atet regulatory expression system for use inStaphylococcus aureus, with the goal of downregulating gene expression via induction of antisense RNA. We demonstrate that this system exhibits a 50- to 100-fold dose-dependent level of induction in bacterial cells grown in culture (i.e., in vitro) and also functions in mice (i.e., in vivo) following oral administration of inducer. To determine whether induced antisense RNA could interfere with chromosomally derived gene expression, we cloned a fragment of theS. aureus alpha-toxin gene (hla) in antisense orientation downstream of the tet promoter system and introduced the construct into S. aureus. Induced antisensehla RNA downregulated chromosomally derived hlagene expression in vitro approximately 14-fold. Similarly, induction ofhla antisense RNA in vivo dramatically reduced alpha-toxin expression in two different murine models of S. aureusinfection. Most importantly, this reduction completely eliminated the lethality of the infection. These results indicate that thetet regulatory system functions efficiently in S. aureus and induced antisense RNA can effectively downregulate chromosomal gene expression both in vitro and in vivo.

scholarly journals An Enzybiotic Regimen for the Treatment of Methicillin-Resistant Staphylococcus aureus Orthopaedic Device-Related Infection

Antibiotics ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1186
Author(s):  
Eric T. Sumrall ◽  
Marloes I. Hofstee ◽  
Daniel Arens ◽  
Christian Röhrig ◽  
Susanne Baertl ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Trauma Surgery ◽  
In Vitro Models ◽  
Fracture Repair ◽  
Combination Regimen ◽  
Bacterial Survival ◽  
Methicillin Resistant ◽  
Orthopaedic Device

Orthopaedic device-related infection (ODRI) presents a significant challenge to the field of orthopaedic and trauma surgery. Despite extensive treatment involving surgical debridement and prolonged antibiotic therapy, outcomes remain poor. This is largely due to the unique abilities of Staphylococcus aureus, the most common causative agent of ODRI, to establish and protect itself within the host by forming biofilms on implanted devices and staphylococcal abscess communities (SACs). There is a need for novel antimicrobials that can readily target such features. Enzybiotics are a class of antimicrobial enzymes derived from bacteria and bacteriophages, which function by enzymatically degrading bacterial polymers essential to bacterial survival or biofilm formation. Here, we apply an enzybiotic-based combination regimen to a set of in vitro models as well as in a murine ODRI model to evaluate their usefulness in eradicating established S. aureus infection, compared to classical antibiotics. We show that two chimeric endolysins previously selected for their functional efficacy in human serum in combination with a polysaccharide depolymerase reduce bacterial CFU numbers 10,000-fold in a peg model and in an implant model of biofilm. The enzyme combination also completely eradicates S. aureus in a SAC in vitro model where classical antibiotics are ineffective. In an in vivo ODRI model in mice, the antibiofilm effects of this enzyme regimen are further enhanced when combined with a classical gentamicin/vancomycin treatment. In a mouse model of methicillin-resistant S. aureus (MRSA) ODRI following a fracture repair, a combined local enzybiotic/antibiotic treatment regimen showed a significant CFU reduction in the device and the surrounding soft tissue, as well as significant prevention of weight loss. These outcomes were superior to treatment with antibiotics alone. Overall, this study demonstrates that the addition of enzybiotics, which are distinguished by their extremely rapid killing efficacy and antibiofilm activities, can enhance the treatment of severe MRSA ODRI.

scholarly journals Mechanisms of fluoroquinolone resistance in genetically related strains of Staphylococcus aureus.

1997 ◽  
Vol 41 (12) ◽  
pp. 2733-2737 ◽  
Author(s):  
G W Kaatz ◽  
S M Seo
Keyword(s):  
Staphylococcus Aureus ◽  
Resistance Mechanisms ◽  
Single Step ◽  
Fluoroquinolone Resistance ◽  
Aureus Strain ◽  
Topoisomerase Iv ◽  
Single Strain ◽  
Derivatives Of

Fluoroquinolone resistance in Staphylococcus aureus results from amino acid substitutions at particular locations in the DNA gyrase A and B subunits as well as in the topoisomerase IV A subunit and from NorA-mediated efflux. More than one resistance mechanism may be present in a single strain. Fluoroquinolone-resistant derivatives of SA-1199, a methicillin-susceptible S. aureus strain, were selected in vivo or in vitro, and their mechanisms of fluoroquinolone resistance were identified. We found that many of the resistance mechanisms described above can develop in derivatives of a single parent strain, either singly or in combination, and can arise in a single step. Variances in MICs for strains with the same apparent resistance mechanisms likely are due to the presence of new or undetected but established means of fluoroquinolone resistance. NorA-mediated resistance can occur in the apparent absence of topoisomerase mutations and in some strains may be the result of a promoter region mutation causing increased expression of norA. However, increased expression of norA can occur independently of this mutation, suggesting that a regulatory locus for this gene exists elsewhere on the chromosome.

scholarly journals Low-Level Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein 1 In Vitro Associated with qacA Gene Carriage Is Independent of Multidrug Efflux Pump Activity

2006 ◽  
Vol 50 (7) ◽  
pp. 2448-2454 ◽  
Author(s):  
A. S. Bayer ◽  
L. I. Kupferwasser ◽  
M. H. Brown ◽  
R. A. Skurray ◽  
S. Grkovic ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cytoplasmic Membrane ◽  
Efflux Pump ◽  
Multidrug Transporter ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Pump Activity ◽  
The Impact

ABSTRACT Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1r) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1r and (ii) tPMP-1r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1r qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1 susceptible (tPMP-1s). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Etr, but not tPMP-1r, in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA-bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1r phenotype. These data suggest that qacA-encoded tPMP-1r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux.

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