Modeling of Effective Antimicrobials to Reduce Staphylococcus aureus Virulence Gene Expression Using a Two-Compartment Hollow Fiber Infection Model

Toxins produced by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) contribute to virulence. We developed a statistical approach to determine an optimum sequence of antimicrobials to treat CA-MRSA infections based on an antimicrobial’s ability to reduce virulence. In an in vitro pharmacodynamic hollow fiber model, expression of six virulence genes (lukSF-PV, sek, seq, ssl8, ear, and lpl10) in CA-MRSA USA300 was measured by RT-PCR at six time points with or without human-simulated, pharmacokinetic dosing of five antimicrobials (clindamycin, minocycline, vancomycin, linezolid, and trimethoprim/sulfamethoxazole (SXT)). Statistical modeling identified the antimicrobial causing the greatest decrease in virulence gene expression at each time-point. The optimum sequence was SXT at T0 and T4, linezolid at T8, and clindamycin at T24–T72 when lukSF-PV was weighted as the most important gene or when all six genes were weighted equally. This changed to SXT at T0–T24, linezolid at T48, and clindamycin at T72 when lukSF-PV was weighted as unimportant. The empirical p-value for each optimum sequence according to the different weights was 0.001, 0.0009, and 0.0018 with 10,000 permutations, respectively, indicating statistical significance. A statistical method integrating data on change in gene expression upon multiple antimicrobial exposures is a promising tool for identifying a sequence of antimicrobials that is effective in sustaining reduced CA-MRSA virulence.

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Role of BrnQ1 and BrnQ2 in Branched-Chain Amino Acid Transport and Virulence in Staphylococcus aureus

Infection and Immunity ◽

10.1128/iai.02542-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 1019-1029 ◽

Cited By ~ 21

Author(s):

Julienne C. Kaiser ◽

Sameha Omer ◽

Jessica R. Sheldon ◽

Ian Welch ◽

David E. Heinrichs

Keyword(s):

Staphylococcus Aureus ◽

Virulence Gene ◽

Defined Medium ◽

Infection Model ◽

Content Type ◽

Virulence Gene Expression ◽

Branched Chain Amino Acid ◽

Branched Chain

The branched-chain amino acids (BCAAs; Ile, Leu, and Val) not only are important nutrients for the growth ofStaphylococcus aureusbut also are corepressors for CodY, which regulates virulence gene expression, implicating BCAAs as an important link between the metabolic state of the cell and virulence. BCAAs are either synthesized intracellularly or acquired from the environment.S. aureusencodes three putative BCAA transporters, designated BrnQ1, BrnQ2, and BrnQ3; their functions have not yet been formally tested. In this study, we mutated all threebrnQparalogs so as to characterize their substrate specificities and their roles in growthin vitroandin vivo. We demonstrated that in the community-associated, methicillin-resistantS. aureus(CA-MRSA) strain USA300, BrnQ1 is involved in uptake of all three BCAAs, BrnQ2 transports Ile, and BrnQ3 does not have a significant role in BCAA transport under the conditions tested. Of the three, only BrnQ1 is essential for USA300 to grow in a chemically defined medium that is limited for Leu or Val. Interestingly, we observed that abrnQ2mutant grew better than USA300 in media limited for Leu and Val, owing to the fact that this mutation leads to overexpression ofbrnQ1. In a murine infection model, thebrnQ1mutant was attenuated, but in contrast,brnQ2mutants had significantly increased virulence compared to that of USA300, a phenotype we suggest is at least partially linked to enhancedin vivoscavenging of Leu and Val through BrnQ1. These data uncover a hitherto-undiscovered connection between nutrient acquisition and virulence in CA-MRSA.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01023-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 241-247 ◽

Cited By ~ 32

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05113-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 140-147 ◽

Cited By ~ 13

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.

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Role of Staphylococcus aureus Global Regulators sae and σB in Virulence Gene Expression during Device-Related Infection

Infection and Immunity ◽

10.1128/iai.73.6.3415-3421.2005 ◽

2005 ◽

Vol 73 (6) ◽

pp. 3415-3421 ◽

Cited By ~ 79

Author(s):

Christiane Goerke ◽

Ursula Fluckiger ◽

Andrea Steinhuber ◽

Vittoria Bisanzio ◽

Martina Ulrich ◽

...

Keyword(s):

Gene Expression ◽

Staphylococcus Aureus ◽

Virulence Gene ◽

Northern Analysis ◽

Virulence Gene Expression ◽

Global Regulators ◽

Reverse Transcription Pcr ◽

Direct Cross

ABSTRACT The ability of Staphylococcus aureus to adapt to different environments is due to a regulatory network comprising several loci. Here we present a detailed study of the interaction between the two global regulators sae and σB of S. aureus and their influence on virulence gene expression in vitro, as well as during device-related infection. The expression of sae, asp23, hla, clfA, coa, and fnbA was determined in strain Newman and its isogenic saeS/R and sigB mutants by Northern analysis and LightCycler reverse transcription-PCR. There was no indication of direct cross talk between the two regulators. sae had a dominant effect on target gene expression during device-related infection. σB seemed to be less active throughout the infection than under induced conditions in vitro.

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Virulence Gene Expression of Staphylococcus aureus in Human Skin

Frontiers in Microbiology ◽

10.3389/fmicb.2021.692023 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ana Rita Cruz ◽

Jos A. G. van Strijp ◽

Fabio Bagnoli ◽

Andrea G. O. Manetti

Keyword(s):

Gene Expression ◽

Staphylococcus Aureus ◽

Human Skin ◽

Time Course ◽

Virulence Gene ◽

Sweat Glands ◽

Infection Model ◽

Related Factors ◽

Virulence Gene Expression ◽

Alpha Hemolysin

Staphylococcus aureus is the main cause of human skin and soft tissue infections. However, S. aureus pathogenicity within the skin is not fully characterized. Here, we implemented an S. aureus cutaneous infection model using human skin explants and performed a time-course infection to study the gene expression profile of a large panel of virulence-related factors of S. aureus USA300 LAC strain, by high-throughput RT-PCR. We pinpointed the genes that were differentially regulated by the bacteria in the skin tissues and identified 12 virulence factors that were upregulated at all time points assessed. Finally, using confocal microscopy, we show that the expression of alpha-hemolysin by S. aureus varies dependent on the skin niche and that the bacteria preferentially accumulates inside sweat glands and ducts. Taken together, our study gives insights about the pathogenic lifestyle of S. aureus within human skin tissues, which may contribute for the development of anti-S. aureus therapeutic strategies.

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Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon

Diseases of Aquatic Organisms ◽

10.3354/dao03475 ◽

2020 ◽

Vol 139 ◽

pp. 153-160

Author(s):

S Peeralil ◽

TC Joseph ◽

V Murugadas ◽

PG Akhilnath ◽

VN Sreejith ◽

...

Keyword(s):

Gene Expression ◽

Virulence Genes ◽

Vibrio Harveyi ◽

Penaeus Monodon ◽

Challenge Test ◽

Virulence Gene ◽

Economic Losses ◽

Virulence Gene Expression

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

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Norlichexanthone Reduces Virulence Gene Expression and Biofilm Formation in Staphylococcus aureus

PLoS ONE ◽

10.1371/journal.pone.0168305 ◽

2016 ◽

Vol 11 (12) ◽

pp. e0168305 ◽

Cited By ~ 24

Author(s):

Mara Baldry ◽

Anita Nielsen ◽

Martin S. Bojer ◽

Yu Zhao ◽

Cathrine Friberg ◽

...

Keyword(s):

Gene Expression ◽

Staphylococcus Aureus ◽

Biofilm Formation ◽

Virulence Gene ◽

Virulence Gene Expression

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Staphylococcus aureus CcpA Affects Virulence Determinant Production and Antibiotic Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.4.1183-1194.2006 ◽

2006 ◽

Vol 50 (4) ◽

pp. 1183-1194 ◽

Cited By ~ 127

Author(s):

Kati Seidl ◽

Martin Stucki ◽

Martin Ruegg ◽

Christiane Goerke ◽

Christiane Wolz ◽

...

Keyword(s):

Gene Expression ◽

Staphylococcus Aureus ◽

Capsular Polysaccharide ◽

Protein A ◽

Virulence Gene ◽

Exponential Growth Phase ◽

Virulence Determinants ◽

Resistance Level ◽

Virulence Gene Expression ◽

Oxacillin Resistance

ABSTRACT Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the ccpA homologue (saCOL1786) in Staphylococcus aureus was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the effect was only transient during the exponential-growth phase as long as glucose was present in the medium. Depletion of glucose and production of lactate was delayed, while the level of excretion of acetate was less affected and was even higher in the mutant culture. On solid medium, in contrast, growth of the ΔccpA mutant resulted in smaller colonies containing a lower number of CFU per colony. Deletion of CcpA had an effect on the expression of important virulence factors of S. aureus by down-regulating RNAIII, the effector molecule of the agr locus, and altering the transcription patterns of hla, encoding α-hemolysin, and spa, encoding protein A. CcpA inactivation markedly reduced the oxacillin resistance levels in the highly methicillin-resistant S. aureus strain COLn and the teicoplanin resistance level in a glycopeptide-intermediate-resistant S. aureus strain. The presence of CcpA in the capsular polysaccharide serotype 5 (CP5)-producing strain Newman abolished capsule formation and decreased cap operon transcription in the presence of glucose. The staphylococcal CcpA thus not only is involved in the regulation of carbon metabolism but seems to function as a modulator of virulence gene expression as well.

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