In Vitro Analysis of the Staphylococcus aureus Lipoteichoic Acid Synthase Enzyme Using Fluorescently Labeled Lipids

ABSTRACT Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria. The key enzyme responsible for polyglycerolphosphate lipoteichoic acid synthesis in the Gram-positive pathogen Staphylococcus aureus is the membrane-embedded lipoteichoic acid synthase enzyme, LtaS. It is presumed that LtaS hydrolyzes the glycerolphosphate head group of the membrane lipid phosphatidylglycerol (PG) and catalyzes the formation of the polyglycerolphosphate LTA backbone chain. Here we describe an in vitro assay for this new class of enzyme using PG with a fluorescently labeled fatty acid chain (NBD-PG) as the substrate and the recombinant soluble C-terminal enzymatic domain of LtaS (eLtaS). Thin-layer chromatography and mass spectrometry analysis of the lipid reaction products revealed that eLtaS is sufficient to cleave the glycerolphosphate head group from NBD-PG, resulting in the formation of NBD-diacylglycerol. An excess of soluble glycerolphosphate could not compete with the hydrolysis of the fluorescently labeled PG lipid substrate, in contrast to the addition of unlabeled PG. This indicates that the enzyme recognizes and binds other parts of the lipid substrate, besides the glycerolphosphate head group. Furthermore, eLtaS activity was Mn2+ ion dependent; Mg2+ and Ca2+ supported only weak enzyme activity. Addition of Zn2+ or EDTA inhibited enzyme activity even in the presence of Mn2+. The pH optimum of the enzyme was 6.5, characteristic for an enzyme that functions extracellularly. Lastly, we show that the in vitro assay can be used to study the enzyme activities of other members of the lipoteichoic acid synthase enzyme family.

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Novel Synthetic (Poly)Glycerolphosphate-Based Antistaphylococcal Conjugate Vaccine

Infection and Immunity ◽

10.1128/iai.00271-13 ◽

2013 ◽

Vol 81 (7) ◽

pp. 2554-2561 ◽

Cited By ~ 13

Author(s):

Quanyi Chen ◽

Jay Dintaman ◽

Andrew Lees ◽

Goutam Sen ◽

David Schwartz ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Conjugate Vaccine ◽

Animal Studies ◽

Lipoteichoic Acid ◽

Gram Positive ◽

Content Type ◽

Cpg Oligodeoxynucleotides ◽

Staphylococcal Infections

ABSTRACTStaphylococcal infections are a major source of global morbidity and mortality. Currently there exists no antistaphylococcal vaccine in clinical use. Previous animal studies suggested a possible role for purified lipoteichoic acid as a vaccine target for eliciting protective IgG to several Gram-positive pathogens. Since the highly conserved (poly)glycerolphosphate backbone of lipoteichoic acid is a major antigenic target of the humoral immune system during staphylococcal infections, we developed a synthetic method for producing glycerol phosphoramidites to create a covalent 10-mer of (poly)glycerolphosphate for potential use in a conjugate vaccine. We initially demonstrated that intactStaphylococcus aureuselicits murine CD4+T cell-dependent (poly)glycerolphosphate-specific IgM and IgG responsesin vivo. Naive mice immunized with a covalent conjugate of (poly)glycerolphosphate and tetanus toxoid in alum plus CpG-oligodeoxynucleotides produced high secondary titers of serum (poly)glycerolphosphate-specific IgG. Sera from immunized mice enhanced opsonophagocytic killing of liveStaphylococcus aureusin vitro. Mice actively immunized with the (poly)glycerolphosphate conjugate vaccine showed rapid clearance of staphylococcal bacteremiain vivorelative to mice similarly immunized with an irrelevant conjugate vaccine. In contrast to purified, natural lipoteichoic acid, the (poly)glycerolphosphate conjugate vaccine itself exhibited no detectable inflammatory activity. These data suggest that a synthetic (poly)glycerolphosphate-based conjugate vaccine will contribute to active protection against extracellular Gram-positive pathogens expressing this highly conserved backbone structure in their membrane-associated lipoteichoic acid.

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In Vitro activity of a Novel Benzoquinolizine Antibiotic, Levonadifloxacin (WCK 771) against Blood Stream Gram-Positive Isolates from a Tertiary Care Hospital

Journal of Laboratory Physicians ◽

10.1055/s-0040-1720944 ◽

2020 ◽

Vol 12 (03) ◽

pp. 230-232

Author(s):

Dhruv Mamtora ◽

Sanjith Saseedharan ◽

Ritika Rampal ◽

Prashant Joshi ◽

Pallavi Bhalekar ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Tertiary Care Hospital ◽

Tertiary Care ◽

Blood Stream ◽

Tolerability Profile ◽

Care Hospital ◽

Coagulase Negative Staphylococci ◽

Gram Positive ◽

Methicillin Resistant

Abstract Background Blood stream infections (BSIs) due to Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) are associated with high mortality ranging from 10 to 60%. The current anti-MRSA agents have limitations with regards to safety and tolerability profile which limits their prolonged usage. Levonadifloxacin and its oral prodrug alalevonadifloxacin, a novel benzoquinolizine antibiotic, have recently been approved for acute bacterial skin and skin structure infections including diabetic foot infections and concurrent bacteremia in India. Methods The present study assessed the potency of levonadifloxacin, a novel benzoquinolizine antibiotic, against Gram-positive blood stream clinical isolates (n = 31) collected from January to June 2019 at a tertiary care hospital in Mumbai, India. The susceptibility of isolates to antibacterial agents was defined following the Clinical and Laboratory Standard Institute interpretive criteria (M100 E29). Results High prevalence of MRSA (62.5%), quinolone-resistant Staphylococcus aureus (QRSA) (87.5%), and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) (82.35%) were observed among bacteremic isolates. Levonadifloxacin demonstrated potent activity against MRSA, QRSA, and MR-CoNS strains with significantly lower minimum inhibitory concentration MIC50/90 values of 0.5/1 mg/L as compared with levofloxacin (8/32 mg/L) and moxifloxacin (2/8 mg/L). Conclusion Potent bactericidal activity coupled with low MICs support usage of levonadifloxacin for the management of BSIs caused by multidrug resistant Gram-positive bacteria.

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In Vitro and In Vivo Activities of DA-7867, a New Oxazolidinone, against Aerobic Gram-Positive Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2498-2500.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2498-2500 ◽

Cited By ~ 7

Author(s):

Eun Jeong Yoon ◽

Yeong Woo Jo ◽

Sung Hak Choi ◽

Tae Ho Lee ◽

Jae Keol Rhee ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Streptococcus Pneumoniae ◽

Gram Positive ◽

Methicillin Resistant ◽

Gram Positive Bacteria ◽

Vancomycin Resistant Enterococci ◽

Infection Models ◽

Vancomycin Resistant

ABSTRACT In vitro and in vivo activities of DA-7867 were assessed against methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and penicillin-resistant Streptococcus pneumoniae. All isolates were inhibited by DA-7867 at ≤0.78 μg/ml, a four-times-lower concentration than that of inhibition by linezolid. For murine infection models, DA-7867 also exhibited greater efficacy than linezolid against all isolates tested.

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A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus

Access Microbiology ◽

10.1099/acmi.ac2020.po0394 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

Shauna D. Drumm ◽

Rebecca Owens ◽

Jennifer Mitchell ◽

Orla M. Keane

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Surface Proteins ◽

Host Cells ◽

Mass Spectrometry Analysis ◽

In Vitro Assays ◽

Immune Recognition ◽

Independent Manner ◽

Genes Encoding

In Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins. Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes. Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages. These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.

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Screening of Escherichia coli Species Biodiversity Reveals New Biofilm-Associated Antiadhesion Polysaccharides

mBio ◽

10.1128/mbio.00043-11 ◽

2011 ◽

Vol 2 (3) ◽

Cited By ~ 55

Author(s):

Olaya Rendueles ◽

Laetitia Travier ◽

Patricia Latour-Lambert ◽

Thierry Fontaine ◽

Julie Magnus ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Bacterial Adhesion ◽

Bioactive Molecules ◽

Bacterial Biofilms ◽

Gram Positive ◽

Content Type ◽

E Coli ◽

Species Biodiversity

ABSTRACTBacterial biofilms often form multispecies communities in which complex but ill-understood competition and cooperation interactions occur. In light of the profound physiological modifications associated with this lifestyle, we hypothesized that the biofilm environment might represent an untapped source of natural bioactive molecules interfering with bacterial adhesion or biofilm formation. We produced cell-free solutions extracted fromin vitromature biofilms formed by 122 naturalEscherichia coliisolates, and we screened these biofilm extracts for antiadhesion molecules active on a panel of Gram-positive and Gram-negative bacteria. Using this approach, we showed that 20% of the tested biofilm extracts contained molecules that antagonize bacterial growth or adhesion. We characterized a compound, produced by a commensal animalE. colistrain, for which activity is detected only in biofilm extract. Biochemical and genetic analyses showed that this compound corresponds to a new type of released high-molecular-weight polysaccharide whose biofilm-associated production is regulated by the RfaH protein. We demonstrated that the antiadhesion activity of this polysaccharide was restricted to Gram-positive bacteria and that its production reduced susceptibility to invasion and provided rapid exclusion ofStaphylococcus aureusfrom mixedE. coliandS. aureusbiofilms. Our results therefore demonstrate that biofilms contain molecules that contribute to the dynamics of mixed bacterial communities and that are not or only poorly detected in unconcentrated planktonic supernatants. Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms on medical devices.IMPORTANCEWe sought to demonstrate that bacterial biofilms are reservoirs for unknown molecules that antagonize bacterial adhesion. The use of natural strains representative ofEscherichia colispecies biodiversity showed that nonbiocidal antiadhesion polysaccharides are frequently found in mature biofilm extracts (bacterium-free suspensions which contain soluble molecules produced within the biofilm). Release of an antiadhesion polysaccharide confers a competitive advantage upon the producing strain against clinically relevant pathogens such asStaphylococcus aureus. Hence, exploring the biofilm environment provides a better understanding of bacterial interactions within complex communities and could lead to improved control of pathogen colonization.

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Nitroxide Functionalized Antibiotics Are Promising Eradication Agents against Staphylococcus aureus Biofilms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01685-19 ◽

2019 ◽

Vol 64 (1) ◽

Cited By ~ 1

Author(s):

Anthony D. Verderosa ◽

Rabeb Dhouib ◽

Kathryn E. Fairfull-Smith ◽

Makrina Totsika

Keyword(s):

Staphylococcus Aureus ◽

Bacterial Biofilms ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Microscopy Analysis ◽

Biofilm Dispersal ◽

Biofilm Structure ◽

Dispersal Activity

ABSTRACT Treatment of biofilm-related Staphylococcus aureus infections represents an important medical challenge worldwide, as biofilms, even those involving drug-susceptible S. aureus strains, are highly refractory to conventional antibiotic therapy. Nitroxides were recently shown to induce the dispersal of Gram-negative biofilms in vitro, but their action against Gram-positive bacterial biofilms remains unknown. Here, we demonstrate that the biofilm dispersal activity of nitroxides extends to S. aureus, a clinically important Gram-positive pathogen. Coadministration of the nitroxide CTEMPO (4-carboxy-2,2,6,6-tetramethylpiperidin-1-yloxyl) with ciprofloxacin significantly improved the biofilm eradication activity of the antibiotic against S. aureus. Moreover, covalently linking the nitroxide to the antibiotic moiety further reduced the ciprofloxacin minimal biofilm eradication concentration. Microscopy analysis revealed that fluorescent nitroxide-antibiotic hybrids could penetrate S. aureus biofilms and enter cells localized at the surface and base of the biofilm structure. No toxicity to human cells was observed for the nitroxide CTEMPO or the nitroxide-antibiotic hybrids. Taken together, our results show that nitroxides can mediate the dispersal of Gram-positive biofilms and that dual-acting biofilm eradication antibiotics may provide broad-spectrum therapies for the treatment of biofilm-related infections.

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In VitroActivities of Tedizolid and Linezolid against Gram-Positive Cocci Associated with Acute Bacterial Skin and Skin Structure Infections and Pneumonia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00390-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6262-6265 ◽

Cited By ~ 24

Author(s):

Ko-Hung Chen ◽

Yu-Tsung Huang ◽

Chun-Hsing Liao ◽

Wang-Hui Sheng ◽

Po-Ren Hsueh

Keyword(s):

Staphylococcus Aureus ◽

Dilution Method ◽

Agar Dilution Method ◽

Gram Positive ◽

Content Type ◽

Skin Structure ◽

Streptococcus Anginosus ◽

Wide Range ◽

Gram Positive Cocci

ABSTRACTTedizolid is a novel, expanded-spectrum oxazolidinone with potent activity against a wide range of Gram-positive pathogens. A total of 425 isolates of Gram-positive bacteria were obtained consecutively from patients with acute bacterial skin and skin structure infections (ABSSSIs) or pneumonia. These isolates included methicillin-susceptibleStaphylococcus aureus(MSSA) (n= 100), methicillin-resistantStaphylococcus aureus(MRSA) (n= 100),Streptococcus pyogenes(n= 50),Streptococcus agalactiae(n= 50),Streptococcus anginosusgroup (n= 75),Enterococcus faecalis(n= 50), and vancomycin-resistant enterococci (VRE) (Enterococcus faecium) (n= 50). The MICs of tedizolid and linezolid were determined by the agar dilution method. Tedizolid exhibited betterin vitroactivities than linezolid against MSSA (MIC90s, 0.5 versus 2 μg/ml), MRSA (MIC90s, 0.5 versus 2 μg/ml),S. pyogenes(MIC90s, 0.5 versus 2 μg/ml),S. agalactiae(MIC90s, 0.5 versus 2 μg/ml),Streptococcus anginosusgroup (MIC90s, 0.5 versus 2 μg/ml),E. faecalis(MIC90s, 0.5 versus 2 μg/ml), and VRE (MIC90s, 0.5 versus 2 μg/ml). The tedizolid MICs againstE. faecalis(n= 3) and VRE (n= 2) intermediate to linezolid (MICs, 4 μg/ml) were 1 μg/ml and 0.5 μg/ml, respectively. The tedizolid MIC90s against S. anginosus,S. constellatus, andS. intermediuswere 0.5, 1, and 0.5 μg/ml, respectively, and the rates of susceptibility based on the U.S. FDA MIC interpretive breakpoints to the isolates were 16%, 28%, and 72%, respectively. Tedizolid exhibited 2- to 4-fold betterin vitroactivities than linezolid against a variety of Gram-positive cocci associated with ABSSSIs and pneumonia. The lower susceptibilities of tedizolid against isolates ofS. anginosusandS. constellatusthan against those ofS. intermediusin Taiwan were noted.

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In vitro activity of the trinem sanfetrinem (GV104326) against gram-positive organisms.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.9.2142 ◽

1996 ◽

Vol 40 (9) ◽

pp. 2142-2146 ◽

Cited By ~ 11

Author(s):

K V Singh ◽

T M Coque ◽

B E Murray

Keyword(s):

Staphylococcus Aureus ◽

Vitro Activity ◽

In Vitro Activity ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Microdilution Method ◽

Broth Microdilution Method ◽

Gentamicin Resistance ◽

High Level

The in vitro activity of the trinem sanfetrinem (formerly GV104326) (GV) was compared with that of vancomycin, ampicillin, and/or nafcillin against 287 gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and multiresistant enterococci, by the agar and microbroth dilution methods. GV demonstrated 2 to 16 times more activity than ampicillin and nafcillin against the majority of these organisms. The MIC range of GV was 16 to 64 micrograms/ml for 19 Enterococcus faecium strains that were highly resistant to ampicillin (ampicillin MIC range, 64 to 512 micrograms/ml) and vancomycin resistant and 0.25 to 32 micrograms/ml for resistant Rhodococcus spp. Similar activities (+/-1 dilution) were observed by either the agar or the broth microdilution method. GV demonstrated bactericidal activity against a beta-lactamase-producing Enterococcus faecalis strain and against two methicillin-susceptible Staphylococcus aureus strains in 10(5)-CFU/ml inocula. Synergy between GV and gentamicin was observed against an E. faecalis strain that lacked high-level gentamicin resistance. The activity of GV suggests this compound warrants further study.

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New Gram-Positive Agents: the Next Generation of Oxazolidinones and Lipoglycopeptides

Journal of Clinical Microbiology ◽

10.1128/jcm.03395-15 ◽

2016 ◽

Vol 54 (9) ◽

pp. 2225-2232 ◽

Cited By ~ 23

Author(s):

Matthew P. Crotty ◽

Tamara Krekel ◽

Carey-Ann D. Burnham ◽

David J. Ritchie

Keyword(s):

Staphylococcus Aureus ◽

Antimicrobial Resistance ◽

Next Generation ◽

Gram Positive ◽

Content Type ◽

Skin Structure ◽

Vancomycin Resistant Enterococci ◽

Vancomycin Resistant ◽

The U.S

The growing problem of antimicrobial resistance among bacterial pathogens, including methicillin-resistantStaphylococcus aureus(MRSA) and vancomycin-resistant enterococci (VRE), has reached a critical state. Tedizolid phosphate, dalbavancin, and oritavancin have recently been approved by the U.S. Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections (ABSSSI) and represent the next generation of oxazolidinones and lipoglycopeptides. All three agents exhibitin vitroactivity and clinical efficacy against MRSA. Tedizolid phosphate and oritavancin demonstratein vitroactivity against VRE. These new Gram-positive agents are reviewed here.

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Burdock (Arctium lappa) Leaf Extracts Increase the In Vitro Antimicrobial Efficacy of Common Antibiotics on Gram-positive and Gram-negative Bacteria

Open Chemistry ◽

10.1515/chem-2017-0012 ◽

2017 ◽

Vol 15 (1) ◽

pp. 92-102 ◽

Cited By ~ 4

Author(s):

Lucia Pirvu ◽

Isabela Nicorescu ◽

Cristina Hlevca ◽

Bujor Albu ◽

Valentin Nicorescu

Keyword(s):

Staphylococcus Aureus ◽

Ethanol Extract ◽

Leaf Extracts ◽

Gram Positive ◽

Polyphenol Compounds ◽

Arctium Lappa ◽

Gram Negative ◽

E Coli ◽

Polar Extracts

AbstractThis work aimed to study the potential effects of four Arctii folium extracts, 5 mg gallic [GAE] acid equivalents per 1 mL sample, on six antibiotics (Ampicillin/AM, Tetracycline/TE, Ciprofloxacin/CIP, Sulfamethoxazole-Trimethoprim/SXT, Chloramphenicol/C and Gentamicin/CN) tested on four Gram-positive (Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, and Staphylococcus epidermidis ATCC 12228) and five Gram-negative (Proteus mirabilis ATCC 29245, Escherichia coli ATCC 35218, E. coli ATCC 11229, E. coli ATCC 8739, and Bacillus cereus ATCC 11778) bacteria. Arctii folium extracts were the whole ethanol extract/W and subsequent ethyl acetate/EA, aqueous/AQ, and chloroform/CHL fractions. Chemical qualitative analysis (HPTLC method) emphasized five main polyphenol compounds in Arctii folium polar extracts: chlorogenic acid (Rf≈0.52/0.55) and its isomer, 1,5-di-O-caffeoylquinic acid (Rf≈0.90/0.92), plus cynarin (Rf≈0.77), hyperoside (Rf≈0.68/0.64) and isoquercitrin (Rf≈0.69/0.71). Microbiological screening indicated Arctii folium polar extracts (AQ and W) efficacy on S. epidermidis ATCC 12228; the MIC values were in the range of common antibiotics, being 32 and 128 μg GAE per mL sample respectively. The unpredictable effects (stimulatory or inhibitory) of Arctii folium extracts in combination with typical antibiotics as well as a potential use of the whole ethanol extract/W for restoring the antimicrobial potency of susceptible antibiotics have also been evidenced.

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