scholarly journals Bactericidal Activity of Gentamicin againstEnterococcus faecalis In Vitro and In Vivo

2000 ◽  
Vol 44 (8) ◽  
pp. 2077-2080 ◽  
Author(s):  
Agnès Lefort ◽  
Michel Arthur ◽  
Louis Garry ◽  
Claude Carbon ◽  
Patrice Courvalin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Bactericidal Activity ◽  
Susceptibility Testing ◽  
Rabbit Model ◽  
Intrinsic Activity ◽  
Resistant Mutants ◽  
Number Of Bacteria

ABSTRACT The activity of gentamicin at various concentrations against two strains of Enterococcus faecalis was investigated in vitro and in a rabbit model of aortic endocarditis. In vitro, gentamicin at 0.5 to 4 times the MIC failed to reduce the number of bacteria at 24 h. Rabbit or human serum dramatically increased gentamicin activity, leading to a ≥3-log10 CFU/ml decrease in bacterial counts when the drug concentration exceeded the MIC. Susceptibility testing in the presence of serum was predictive of in vivo activity, since gentamicin alone significantly reduced the number of surviving bacteria in the vegetations if the peak-to-MIC ratio was greater than 1. However, gentamicin selected resistant mutants in rabbits. The intrinsic activity of gentamicin should be taken into account in evaluation of combinations of gentamicin and cell wall-active agents against enterococci.

scholarly journals Activity of LY333328 Combined with Gentamicin In Vitro and in Rabbit Experimental Endocarditis Due to Vancomycin-Susceptible or -Resistant Enterococcus faecalis

2000 ◽  
Vol 44 (11) ◽  
pp. 3017-3021 ◽  
Author(s):  
Agnès Lefort ◽  
Azzam Saleh-Mghir ◽  
Louis Garry ◽  
Claude Carbon ◽  
Bruno Fantin
Keyword(s):  
Enterococcus Faecalis ◽  
Bactericidal Activity ◽  
Type Strain ◽  
Rabbit Model ◽  
Serum Levels ◽  
Intravenous Treatment ◽  
Trough Serum ◽  
Type Strains

ABSTRACT We investigated the activity of LY333328 alone and combined with gentamicin, both in vitro and in a rabbit model of experimental endocarditis, against the susceptible strain Enterococcus faecalis JH2-2 and its two glycopeptide-resistant transconjugants, BM4316 (VanA) and BM4275 (VanB). MICs of LY333328 and gentamicin were 2 and 16 μg/ml, respectively, for the three strains. In vitro, LY333328 alone was bactericidal at 24 h against JH2-2 at a concentration of 2 μg/ml and against BM4316 and BM4275 at a concentration of 30 μg/ml. The combination of LY333328 and gentamicin (4 μg/ml) was synergistic and bactericidal after 24 h of incubation against the three strains at LY333328 concentrations of 2 μg/ml for JH2-2 and 8 μg/ml for BM4275 and BM4316. The combination of LY333328 and gentamicin was the only regimen demonstrating in vitro bactericidal activity against BM4316. In vivo, intravenous treatment with LY333328 alone, providing peak and trough serum levels of 83.3 ± 1.3 and 3.8 ± 0.2 μg/ml, respectively, was inactive against BM4316 and BM4275 and selected mutants resistant to LY333328 in half of the rabbits infected with the VanA-type strain (MICs, 8 to 20 μg/ml). However, the LY333328-gentamicin combination was active against the three strains and prevented the emergence of mutants resistant to both components of the combination. We conclude that the LY333328-gentamicin combination might be of interest for the treatment of enterococcal infections, particularly against VanA-type strains.

scholarly journals Two-Step Acquisition of Resistance to the Teicoplanin-Gentamicin Combination by VanB-Type Enterococcus faecalis In Vitro and in Experimental Endocarditis

1999 ◽  
Vol 43 (3) ◽  
pp. 476-482 ◽  
Author(s):  
Agnès Lefort ◽  
Marina Baptista ◽  
Bruno Fantin ◽  
Florence Depardieu ◽  
Michel Arthur ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Acquired Resistance ◽  
Simultaneous Acquisition ◽  
Resistant Mutants

ABSTRACT The activity of vancomycin and teicoplanin combined with gentamicin was investigated in vitro against strains of Enterococcus faecalis resistant to vancomycin and susceptible to teicoplanin (VanB type) and against mutants that had acquired resistance to teicoplanin by three different mechanisms. In vitro, gentamicin selected mutants with two- to sixfold increases in the level of resistance to this antibiotic at frequencies of 10−6 to 10−7. Teicoplanin selected teicoplanin-resistant mutants at similar frequencies. Both mutations were required to abolish the activity of the gentamicin-teicoplanin combination. As expected, simultaneous acquisition of the two types of mutations was not observed. In therapy with gentamicin or teicoplanin alone, each selected mutants in three of seven rabbits with aortic endocarditis due to VanB-type E. faecalis BM4275. The vancomycin-gentamicin combination selected mutants that were resistant to gentamicin and to the combination. In contrast, the teicoplanin-gentamicin regimen prevented the emergence of mutants resistant to one or both components of the combination. These results suggest that two mutations are also required to suppress the in vivo activity of the teicoplanin-gentamicin combination.

THE MODE OF ACTION OF THE ANTIBIOTIC PAROMOMYCIN ON STAPHYLOCOCCUS AUREUS

1966 ◽  
Vol 12 (6) ◽  
pp. 1157-1165 ◽  
Author(s):  
A. von Seefried ◽  
D. C. Jordan
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Binding Sites ◽  
Bactericidal Action ◽  
Plate Method ◽  
Vitro Binding ◽  
Resistant Mutants ◽  
Intracellular Binding

Paromomycin (Humatin, Parke Davis & Co.), a broad-spectrum aminoglycosidic antibiotic, inhibits the incorporation of amino acids into the trypsinsoluble protein fraction of Staphylococcus aureus 257. Protein synthesis is inhibited immediately, but the synthesis of cell-wall mucopeptide and alcohol-soluble proteins and lipids is not affected for approximately 35 min after antibiotic addition to actively growing cells. Paromomycin, at the ribosomal level, prevents the attachment of amino acyl-s-RNA and causes accumulation of m-RNA.Divalent cations (Ca++ and Mg++) antagonize the bactericidal action of paromomycin and interfere with the in vivo binding of the antibiotic on both the cell surface and the intracellular binding sites. In vitro binding to free ribosomes can be prevented and reversed by both monovalent and divalent cations.Using a "cylinder-plate" method, involving the displacement of antibiotic from cellular fractions by 0.2 M MgCl2, the antibiotic can be recovered from the ribosomes, cytoplasm, and the cell wall of paromomycin-sensitive S. aureus cells, but is not found in any of these fractions isolated from paromomycin-resistant cells developed from the sensitive parent strain. The resistant mutants apparently have lost the ability to adsorb and transport the antibiotic into the cell.

scholarly journals Antibodies to a Surface-Exposed, N-terminal Domain of Aggregation Substance Are Not Protective in the Rabbit Model of Enterococcus faecalis Infective Endocarditis

2001 ◽  
Vol 69 (5) ◽  
pp. 3305-3314 ◽  
Author(s):  
John K. McCormick ◽  
Helmut Hirt ◽  
Christopher M. Waters ◽  
Timothy J. Tripp ◽  
Gary M. Dunny ◽  
...  
Keyword(s):  
Infective Endocarditis ◽  
Enterococcus Faecalis ◽  
Rabbit Model ◽  
Surface Protein ◽  
Conjugative Plasmid ◽  
Aggregation Substance ◽  
Exposed Region ◽  
Correct Sequence

ABSTRACT The aggregation substance (AS) surface protein fromEnterococcus faecalis has been implicated as an important virulence factor for the development of infective endocarditis. To evaluate the role of antibodies specific for Asc10 (the AS protein from the conjugative plasmid pCF10) in protective immunity to infective endocarditis, an N-terminal region of Asc10 lacking the signal peptide and predicted to be surface exposed (amino acids 44 to 331; AS44–331) was cloned with a C-terminal histidine tag translational fusion and expressed fromEscherichia coli. N-terminal amino acid sequencing of the purified protein revealed the correct sequence, and rabbit polyclonal antisera raised against AS44–331 reacted specifically to Asc10 expressed from E. faecalis OG1SSp, but not to other proteins as judged by Western blot analysis. Using these antisera, flow cytometry analysis demonstrated that antibodies to AS44–331 bound to a surface-exposed region of Asc10. Furthermore, antibodies specific for AS44–331were opsonic for E. faecalis expressing Asc10 in vitro but not for cells that did not express Asc10. New Zealand White rabbits immunized with AS44–331 were challenged intravenously withE. faecalis cells constitutively expressing Asc10 in the rabbit model of experimental endocarditis. Highly immune animals did not show significant differences in clearance of organisms from the blood or spleen or in formation of vegetations on the aortic valve, in comparison with nonimmune animals. Although in vivo expression of Asc10 was demonstrated by immunohistochemistry, these experiments provide evidence that immunity to Asc10 does not play a role in protection from experimental infective endocarditis due toE. faecalis and may have important implications for the development of immunological approaches to combat enterococcal endocarditis.

scholarly journals Use of Recombinase-BasedIn VivoExpression Technology To Characterize Enterococcus faecalis Gene Expression during Infection IdentifiesIn Vivo-Expressed Antisense RNAs and Implicates the Protease Eep in Pathogenesis

2011 ◽  
Vol 80 (2) ◽  
pp. 539-549 ◽  
Author(s):  
Kristi L. Frank ◽  
Aaron M. T. Barnes ◽  
Suzanne M. Grindle ◽  
Dawn A. Manias ◽  
Patrick M. Schlievert ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Rabbit Model ◽  
Microscopic Analysis ◽  
Mammalian Host ◽  
Wild Type ◽  
Antisense Transcripts ◽  
Content Type ◽  
Antisense Rnas

ABSTRACTEnterococcus faecalisis a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades.E. faecalismust be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-basedin vivoexpression technology (RIVET) to identify promoters on theE. faecalisOG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putativein vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected inin vitro- andin vivo-grown cells, providing the first evidence ofin vivo-expressed antisense RNAs inE. faecalis. Deletions in thein vivo-activated genes that encode glutamate 5-kinase (proB[EF0038]), the transcriptional regulator EbrA (ebrA[EF1809]), and the membrane metalloprotease Eep (eep[EF2380]) did not hinder biofilm formation inin vitroassays. In a rabbit model of endocarditis, the ΔebrAstrain was fully virulent, the ΔproBstrain was slightly attenuated, and the Δeepstrain was severely attenuated. The Δeepvirulence defect could be complemented by the expression of the wild-type gene intrans. Microscopic analysis of early Δeepbiofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.

scholarly journals In Vitro and In Vivo Activities of PD 0305970 and PD 0326448, New Bacterial Gyrase/Topoisomerase Inhibitors with Potent Antibacterial Activities versus Multidrug-Resistant Gram-Positive and Fastidious Organism Groups

2007 ◽  
Vol 51 (4) ◽  
pp. 1191-1201 ◽  
Author(s):  
Michael D. Huband ◽  
Michael A. Cohen ◽  
Margaret Zurack ◽  
Debra L. Hanna ◽  
Laura A. Skerlos ◽  
...  
Keyword(s):  
Bactericidal Activity ◽  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Multidrug Resistant ◽  
Infection Model ◽  
Topoisomerase Inhibitors ◽  
Gram Positive ◽  
Resistant Mutants

ABSTRACT PD 0305970 and PD 0326448 are new bacterial gyrase and topoisomerase inhibitors (quinazoline-2,4-diones) that possess outstanding in vitro and in vivo activities against a wide spectrum of bacterial species including quinolone- and multidrug-resistant gram-positive and fastidious organism groups. The respective MICs (μg/ml) for PD 0305970 capable of inhibiting ≥90% of bacterial strains tested ranged from 0.125 to 0.5 versus staphylococci, 0.03 to 0.06 versus streptococci, 0.25 to 2 versus enterococci, and 0.25 to 0.5 versus Moraxella catarrhalis, Haemophilus influenzae, Listeria monocytogenes, Legionella pneumophila, and Neisseria spp. PD 0326448 MIC90s were generally twofold higher versus these same organism groups. Comparative quinolone MIC90 values were 4- to 512-fold higher than those of PD 0305970. In testing for frequency of resistance, PD 0305970 and levofloxacin showed low levels of development of spontaneous resistant mutants versus both Staphylococcus aureus and Streptococcus pneumoniae. Unlike quinolones, which target primarily gyrA and parC, analysis of resistant mutants in S. pneumoniae indicates that the likely targets of PD 0305970 are gyrB and parE. PD 0305970 demonstrated rapid bactericidal activity by in vitro time-kill testing versus streptococci. This bactericidal activity carried over to in vivo testing, where PD 0305970 and PD 0326448 displayed outstanding Streptococcus pyogenes 50% protective doses (PD50s) (oral dosing) of 0.7 and 3.6 mg/kg, respectively (ciprofloxacin and levofloxacin PD50s were >100 and 17.7 mg/kg, respectively). PD 0305970 was also potent in a pneumococcal pneumonia mouse infection model (PD50 = 3.2 mg/kg) and was 22-fold more potent than levofloxacin.

scholarly journals Mechanism and Suppression of Lysostaphin Resistance in Oxacillin-Resistant Staphylococcus aureus

2001 ◽  
Vol 45 (5) ◽  
pp. 1431-1437 ◽  
Author(s):  
Michael W. Climo ◽  
Kerstin Ehlert ◽  
Gordon L. Archer
Keyword(s):  
Staphylococcus Aureus ◽  
Aortic Valve ◽  
Rabbit Model ◽  
In Vivo Model ◽  
Nucleotide Sequence Analysis ◽  
Cross Bridge ◽  
Oxacillin Resistance ◽  
Resistant Mutants

ABSTRACT The potential for the development of resistance in oxacillin-resistant Staphylococcus aureus (ORSA) to lysostaphin, a glycylglycine endopeptidase produced byStaphylococcus simulans biovar staphylolyticus, was examined in vitro and in an in vivo model of infection. Following in vitro exposure of ORSA to subinhibitory concentrations of lysostaphin, lysostaphin-resistant mutants were idenitifed among all isolates examined. Resistance to lysostaphin was associated with a loss of resistance to β-lactams and a change in the muropeptide interpeptide cross bridge from pentaglycine to a single glycine. Mutations in femA, the gene required for incorporation of the second and third glycines into the cross bridge, were found following PCR amplification and nucleotide sequence analysis. Complementation of lysostaphin-resistant mutants with pBBB31, which encodes femA, restored the phenotype of oxacillin resistance and lysostaphin susceptibility. Addition of β-lactam antibiotics to lysostaphin in vitro prevented the development of lysostaphin-resistant mutants. In the rabbit model of experimental endocarditis, administration of a low dose of lysostaphin for 3 days led predictably to the appearance of lysostaphin-resistant ORSA mutants in vegetations. Coadministration of nafcillin with lysostaphin prevented the emergence of lysostaphin-resistant mutants and led to a mean reduction in aortic valve vegetation counts of 7.5 log10 CFU/g compared to those for untreated controls and eliminated the isolation of lysostaphin-resistant mutants from aortic valve vegetations. Treatment with nafcillin and lysostaphin given alone led to mean reductions of 1.35 and 1.65 log10 CFU/g respectively. In ORSA, resistance to lysostaphin was associated with mutations in femA, but resistance could be suppressed by the coadministration of β-lactam antibiotics.

scholarly journals Successful Therapy of Experimental Endocarditis Caused by Vancomycin-Resistant Staphylococcus aureus with a Combination of Vancomycin and β-Lactam Antibiotics

2006 ◽  
Vol 50 (9) ◽  
pp. 2951-2956 ◽  
Author(s):  
Paige M. Fox ◽  
Russell J. Lampen ◽  
Katrina S. Stumpf ◽  
Gordon L. Archer ◽  
Michael W. Climo
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Rabbit Model ◽  
Bloodstream Infections ◽  
Aberrant Cell ◽  
Trial Treatment ◽  
Vancomycin Resistant ◽  
Vancomycin Treatment

ABSTRACT VRS1 is the first isolated strain of vancomycin-resistant Staphylococcus aureus (VRSA) found to carry the vanA gene complex previously described in Enterococcus. Under vancomycin pressure, VRS1 makes aberrant cell walls consisting of stem tetrapeptide and depsipeptide that lack the terminal d-Ala-d-Ala residues targeted by vancomycin. Previous data have suggested that this aberrant cell wall is not cross-linked by PBP2a, the enzyme responsible for cell wall transpeptidation in the presence of β-lactam antibiotics. We examined the efficacy of treating VRS1 with a combination of vancomycin and β-lactam antibiotics in vitro and in vivo. We found that the MIC of oxacillin for VRS1 decreased from >256 μg/ml to <1 μg/ml in the presence of vancomycin. Using the rabbit model of endocarditis, we treated VRS1-infected rabbits with nafcillin alone, vancomycin alone, or a combination of nafcillin and vancomycin. Treatment with nafcillin in combination with vancomycin cleared bloodstream infections within 24 h and sterilized 12/13 spleens (92%), as well as 8/13 kidneys (62%), following 3 days of treatment. Mean aortic valve vegetation counts were reduced 3.48 log10 CFU/g with the combination therapy (compared to untreated controls) and were significantly lower than with either vancomycin or nafcillin given alone. VRS1 was extremely virulent in this model, as no untreated rabbits survived the 3-day trial. Treatment of clinical infections due to VRSA with the combination of vancomycin and β-lactams may be an option, based on these results.

scholarly journals Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis

2021 ◽  
Author(s):  
Pei Yi Choo ◽  
Charles Wang ◽  
Michael VanNieuwenhze ◽  
Kimberly Kline
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Enterococcus Faecalis ◽  
General Rule ◽  
Cell Wall Synthesis ◽  
Lipid Ii ◽  
Growing Cell ◽  
Temporal Localization

Enterococcus faecalis relies upon a number of cell wall-associated proteins for virulence. One virulence factor is the sortase-assembled endocarditis and biofilm associated pilus (Ebp), an important factor for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that the pilus sortase covalently links pilus monomers prior to recognition, while the housekeeping sortase cleaves at the LPXTG motif within the terminal pilin subunit, and subsequently attaches assembled pilus fiber to the growing cell wall at sites of new cell wall synthesis. While the cell wall anchoring mechanism and polymerization of Ebp is well characterized, less is known about the spatial and temporal deposition of this protein on the cell surface. We followed the distribution of Ebp and peptidoglycan (PG) throughout the E. faecalis cell cycle via immunofluorescence microscopy and fluorescent D-amino acids (FDAA) staining. Surprisingly, cell surface Ebp did not co-localize with newly synthesized PG. Instead, surface-anchored Ebp was localized to the cell hemisphere but never at the septum where new cell wall is deposited. In addition, the older hemisphere of the E. faecalis diplococcus were completely saturated with Ebp, while Ebp appeared as two foci directly adjacent to the nascent septum in the newer hemisphere. A similar localization pattern was observed for another cell wall anchored substrate by sortase A, aggregation substance (AS), suggesting that this may be a general rule for all SrtA substrates in E. faecalis. When cell wall synthesis was inhibited by ramoplanin, an antibiotic that binds and sequesters lipid II cell wall precursors, new Ebp deposition at the cell surface was not disrupted. These data suggest an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto un-crosslinked cell wall, independent of new PG synthesis.

scholarly journals Influence of Resistance to Streptogramin A Type Antibiotics on the Activity of Quinupristin-Dalfopristin In Vitro and in Experimental Endocarditis Due toStaphylococcus aureus

2000 ◽  
Vol 44 (5) ◽  
pp. 1168-1173 ◽  
Author(s):  
Virginie Zarrouk ◽  
Bülent Bozdogan ◽  
Roland Leclercq ◽  
Louis Garry ◽  
Claude Carbon ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Body Weight ◽  
Bactericidal Activity ◽  
Resistant Bacteria ◽  
Clinical Strains ◽  
Resistant Mutants ◽  
Reduced Activity

ABSTRACT We evaluated the activity of quinupristin-dalfopristin (Q-D) against three clinical strains of Staphylococcus aureussusceptible to Q (MIC, 8 μg/ml) and Q-D (MICs, 0.5 to 1 μg/ml) but displaying various levels of susceptibility to D. D was active against S. aureus HM 1054 (MIC, 4 μg/ml) and had reduced activity against S. aureus RP 13 and S. aureus N 95 (MICs, 32 and 64 μg/ml, respectively). In vitro, Q-D at a concentration two times the MIC (2×MIC) produced reductions of 4.3, 3.9, and 5.8 log10 CFU/ml after 24 h of incubation for HM 1054, RP 13, and N 95, respectively. Comparable killing was obtained at 8×MIC. Q-D-resistant mutants were selected in vitro at a frequency of 2 × 10−8 to 2 × 10−7 for the three strains on agar containing 2×MIC of Q-D; no resistant bacteria were detected at 4×MIC. Rabbits with aortic endocarditis were treated for 4 days with Q-D at 30 mg/kg of body weight intramuscularly (i.m.) three times a day (t.i.d.) or vancomycin at 50 mg/kg i.m. t.i.d. In vivo, Q-D and vancomycin were similarly active and bactericidal against the three tested strains compared to the results for control animals (P < 0.01). Among animals infected with RP 13 and treated with Q-D, one rabbit retained Q-D-resistant mutants that were resistant to Q and to high levels of D (MICs, 64, >256, and 8 μg/ml for Q, D, and Q-D, respectively). We conclude that the bactericidal activity of Q-D against strains with reduced susceptibility to D and susceptible to Q-D is retained and is comparable to that of vancomycin. Acquisition of resistance to both Q and D is necessary to select resistance to Q-D.

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