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PubMed2026

Genomic insights into an optrA-carrying plasmid associated with linezolid resistance in clinical Enterococcus faecalis isolates, Argentina.

The spread of the transferable optrA gene poses an increasing threat to the clinical efficacy of oxazolidinones. Here, we characterized a novel optrA-carrying plasmid, pEfa-optrA-Arg, from a linezolid-resistant Enterococcus faecalis clinical isolate from Argentina. The 68,653-bp conjugative plasmid harbored optrA together with multiple antimicrobial resistance genes and showed high similarity to a plasmid previously identified in a bovine isolate from Switzerland. pEfa-optrA-Arg, or a closely related variant, was also detected in E. faecalis isolates from several Argentinian hospitals, highlighting the role of horizontal gene transfer in the spread of antimicrobial resistance across human and animal reservoirs within the One Health continuum.

Enterococcus faecalis↗

PubMed1998

Enterococcus faecalis localization in experimental endophthalmitis: role of plasmid-encoded aggregation substance.

Enterococci have emerged as leading agents of nosocomial infection, yet relatively little is known about the pathogenesis of enterococcal disease. In previous studies, we developed an Enterococcus faecalis endophthalmitis infection model which provides unique opportunities to study the evolution of enterococcal disease by direct observation, as well as through sensitive electrophysiologic measures of organ function. The present study was designed to determine whether E. faecalis possesses traits that permit its attachment to mammalian tissues during infection. It was also of interest to determine whether a plasmid-encoded adhesin, aggregation substance, contributes to enterococcal localization or otherwise mediates adherence to alternate sites. These studies found that, in this model, enterococci attach to membranous structures occurring within the vitreous but that this attachment or the course or severity of disease is unaffected by the aggregation substance phenotype.

Adhesins, Bacterial↗

PubMed1990

Serological response in Enterococcus faecalis endocarditis determined by enzyme-linked immunosorbent assay.

Enterococcus (Streptococcus) faecalis expresses three species-specific surface protein antigens of molecular weights 73,000, 40,000, and 37,000. On Western blotting (immunoblotting), they were detected strongly by immunoglobulin G (IgG) in sera from patients with E. faecalis endocarditis, but not in sera from patients with other E. faecalis infections or with endocarditis due to other streptococci. We developed an enzyme-linked immunosorbent assay system to measure IgG, IgM, and IgA levels to these antigens and evaluated its potential as a serodiagnostic test for E. faecalis endocarditis. The test correctly diagnosed E. faecalis endocarditis in 15 of 16 cases. Of 10 cases of endocarditis due to other streptococci and 10 E. faecalis infections other than endocarditis, 9 and 8, respectively, gave negative results. The test should prove particularly useful in culture-negative cases, for which choice of appropriate antibiotic therapy for E. faecalis endocarditis is vital.

Antibodies, Bacterial↗

PubMed1999

Humoral immunity to commensal oral bacteria in human infants: salivary secretory immunoglobulin A antibodies reactive with Streptococcus mitis biovar 1, Streptococcus oralis, Streptococcus mutans, and Enterococcus faecalis during the first two years of life.

Secretory immunoglobulin A (SIgA) antibodies reactive with the pioneer oral streptococci Streptococcus mitis biovar 1 and Streptococcus oralis, the late oral colonizer Streptococcus mutans, and the pioneer enteric bacterium Enterococcus faecalis in saliva samples from 10 human infants from birth to age 2 years were analyzed. Low levels of salivary SIgA1 and SIgA2 antibodies reactive with whole cells of all four species were detected within the first month after birth, even though S. mutans and E. faecalis were not recovered from the mouths of the infants during the study period. Although there was a fivefold increase in the concentration of SIgA between birth and age 2 years, there were no differences between the concentrations of SIgA1 and SIgA2 antibodies reactive with the four species over this time period. When the concentrations of SIgA1 and SIgA2 antibodies reactive with all four species were normalized to the concentrations of SIgA1 and SIgA2 in saliva, SIgA1 and SIgA2 antibodies reactive with these bacteria showed a significant decrease from birth to 2 years of age. Adsorption of each infant's saliva with cells of one species produced a dramatic reduction of antibodies recognizing the other three species. Sequential adsorption of saliva samples removed all SIgA antibody to the bacteria, indicating that the SIgA antibodies were directed to antigens shared by all four species. The induction by the host of a limited immune response to common antigens that are likely not involved in adherence may be among the mechanisms that commensal streptococci employ to persist in the oral cavity.

Antibodies, Bacterial↗

PubMed1995

Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions.

The prgB gene encodes the surface protein Asc10, which mediates cell aggregation resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline resistance plasmid pCF10 in Enterococcus faecalis. Previous Tn5 insertional mutagenesis and sequencing analysis of a 12-kb fragment of pCF10 indicated that a region containing prgX, -Q, -R, -S, and -T, located 3 to 6 kb upstream of prgB, is required to activate the expression of prgB. Complementation studies showed that the positive regulatory region functions in cis in an orientation-dependent manner (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). In order to determine the involvement of each gene in the activation of prgB, Tn5 insertional mutagenesis and exonuclease III deletion analyses of the regulatory region were carried out. The results indicate that prgQ and -S are required for the expression of prgB, while prgX, -R, and -T are not required. Western blot (immunoblot) analysis of these mutants shows that prgQ is also essential for the expression of prgA (encoding the surface exclusion protein Sec10), which is located between prgB and the positive-control region. Complementation analysis demonstrates that a cis-acting regulatory element is located in the prgQ region and that pCF10 sequences in an untranslated region 3' from prgQ are an essential component of the positive-control system. Analyses of various Tn5 insertions in pCF10 genes suggest that transcription reading into this transposon is terminated in E. faecalis but that outward-reading transcripts may initiate from within the ends of Tn5 or from the junction sequences.

Amino Acid Sequence↗

PubMed2006

Occurrence of aminoglycoside-modifying-enzyme genes aac(6')-aph(2"), aph(3'), ant(4') and ant(6) in clinical isolates of Enterococcus faecalis resistant to high-level of gentamicin and amikacin.

The genes coding for 4 aminoglycoside-modifying enzymes AAC(6')-APH(2"), APH(3'), ANT(4') and ANT(6) were determined in 44 Slovak clinical isolates of Enterococcus faecalis with high-level resistance to gentamicin (HLGR, collection 1) and 48 E. faecalis isolates with resistance to amikacin (AR, collection 2). The occurrence of spotted genes was (collection 1 vs. collection 2): aac(6)-aph(2") 81.8 vs. 8.3 %, ant(4') 52.3 vs. 81.3 %, aph(3') 50 vs. 56.3 % and ant(6) 6.8 vs. 4.2 %, the most frequent combinations of genes in the HLGR collection were aac(6')-aph(2") + ant(4') and aac(6')-aph(2") + aph(3). In contrast, the aph(3') + ant(4') gene profile was predominant in AR isolates. None of the isolates contained all four AGME genes simultaneously.

Acetyltransferases↗

PubMed1996

Efficacy of vancomycin and teicoplanin alone and in combination with streptomycin in experimental, low-level vancomycin-resistant, VanB-type Enterococcus faecalis endocarditis.

The efficacy of vancomycin (VM) and teicoplanin (TE), alone and in combination with streptomycin (SM), against enterococci that express low-level VanB-type VM resistance was investigated in experimental endocarditis using isogenic strains of Enterococcus faecalis susceptible to glycopeptides and aminoglycosides or inducibly resistant to low levels of VM (MIC = 16 micrograms/ml). VM was significantly less active against the resistant strain than against the susceptible strain, establishing that low-level VanB-type VM resistance can influence therapeutic efficacy. By contrast, TE had equally good activity against both strains. VM or TE combined with SM was synergistic and bactericidal against the resistant strain in vitro. While both combinations were efficient in reducing bacterial density in vivo, TE plus SM was significantly superior to VM plus SM if valve sterilization was considered. These data suggest that despite the presence of low-level VanB-type resistance, combination therapy with a glycopeptide and SM (and presumably other aminoglycosides to which there is not high-level resistance) will nevertheless provide effective bactericidal activity.

Animals↗

PubMed1989

Scanning electronmicroscopy of Staphylococcus aureus and Enterococcus faecalis exposed to daptomycin.

The novel lipopeptide antibiotic, daptomycin, at a concentration of 8 mg/L, caused gross morphological changes in both a methicillin-sensitive and a methicillin-resistant strain of Staphylococcus aureus and in a strain of Enterococcus faecalis. The earliest (after 1 h) surface lesion observed was the appearance of boss-like processes randomly distributed on the cell surface. Later, grossly deformed bacteria were seen and in two of the three bacteria prolonged exposure led to degeneration of the cells into an amorphous syncytial mass. Omission of calcium (which is known to potentiate the activity of daptomycin) from the culture medium did not affect the morphological response to an inhibitory concentration of the antibiotic.

Anti-Bacterial Agents↗

PubMed2004

Serological and genetic diversity of capsular polysaccharides in Enterococcus faecalis.

Enterococci possess capsular carbohydrate antigens that are targets of opsonic antibodies. These antigens may be used to develop alternative options for the treatment and prevention of enterococcal infections. The present study was done to analyze the diversity of capsular polysaccharides in Enterococcus faecalis. Four type-specific sera were used in an enzyme-linked immunosorbent assay format to detect polysaccharide antigen extracted from bacterial cell walls. A total of 55% of a collection of 29 E. faecalis strains could be grouped into one of four serogroups. Additional analysis of the strains by opsonophagocytic assays revealed agreement between the results of the two methods for 72% of the isolates. An additional four strains could be assigned to a serogroup on the basis of opsonic killing by sera with antibodies against the four prototypes strains, provisionally named CPS-A to CPS-D. The results of the two methods disagreed for one strain (4%). When the results of both methods were combined, 66% of the strains could be classified. One strain had to be assigned to two serogroups. The assignments to the four serogroups were confirmed by analysis of the genetic organization of the biosynthetic capsular polysaccharide (cps) locus. All strains grouped into serotypes CPS-A and CPS-B possessed only the cpsA and cpsB genes, while all strains grouped into serogroups CPS-C and CPS-D possessed an additional eight or nine genes. Our results suggest the existence of a limited number of E. faecalis capsule serotypes, and we provisionally propose four serotypes, named CPS-A to CPS-D, and the respective prototype strains for these families.

Animals↗

PubMed2000

The rpoN gene of Enterococcus faecalis directs sensitivity to subclass IIa bacteriocins.

The final sigma 54 factor has been previously described to be involved in Listeria monocytogenes sensitivity to mesentericin Y105, a subclass IIa bacteriocin. Here, we identified the rpoN gene, encoding final sigma 54, of Enterococcus faecalis JH2-2 and showed that its interruption leads to E. faecalis resistance to different subclass IIa bacteriocins. Moreover, this rpoN mutant remained sensitive to nisin, a class I bacteriocin, suggesting that final sigma 54 is especially involved in sensitivity to subclass IIa bacteriocins.

Bacteriocins↗

PubMed1998

A new theta-type thermosensitive replicon from Lactococcus lactis as an integration vector for Enterococcus faecalis.

We isolated a replication thermosensitive mutant of the theta-type lactococcal pUCL22 replicon. An improved version of this thermosensitive replicon was obtained by fusioning the replication repA gene with the downstream repB gene. The resulting plasmid was named pUCB3522Ts. It is highly instable at 42 degrees C in Enterococcus faecalis. Integration into the chromosome via homologous recombination was monitored using the npr gene of E. faecalis JH2-2 as a target. A 513 bp PCR amplification product from an internal region of this npr gene was cloned into pUCB3522Ts. Integration of this construction into the JH2-2 npr gene was selected by shift temperature, from 30 degrees C to 42 degrees C. 85% of the analysed clones showed integration into the npr gene, demonstrating the practicality of this thermosensitive replicon as a genetic integrative tool for E. faecalis.

Amino Acid Sequence↗

PubMed2002

Molecular typing of selected Enterococcus faecalis isolates: pilot study using multilocus sequence typing and pulsed-field gel electrophoresis.

The present study compared the recently developed multilocus sequence typing (MLST) approach with a well-established molecular typing technique, pulsed-field gel electrophoresis (PFGE), for subspecies differentiation of Enterococcus faecalis isolates. We sequenced intragenic regions of three E. faecalis antigen-encoding genes (ace, encoding a collagen and laminin adhesin; efaA, encoding an endocarditis antigen; and salA, encoding a cell wall associated antigen) and one housekeeping gene (pyrC) of 22 E. faecalis isolates chosen largely for their temporal and geographical diversity, but also including some outbreak isolates. MLST analysis of polymorphic regions of these four genes identified 13 distinct sequence types (STs) with different allelic profiles; the composite sequences generated from the four sequenced gene fragments of individual isolates showed 98.3 to 100% identity among the 22 isolates. We also found that the allelic profiles from two sequences, ace and salA, were sufficient to distinguish all 13 STs of this study. The 13 STs corresponded to 12 different PFGE types, with one previously designated PFGE clone (a widespread U.S. clone of beta-lactamase-producing isolates) being classified into two highly related STs which differed at 2 of 2,894 bases, both in the same allele. MLST also confirmed the clonal relationships among the isolates of two other PFGE clonal groups, including vancomycin resistant isolates. Thus, this pilot study with representative E. faecalis isolates suggests that, similar to PFGE, the sequence-based typing method may be useful for differentiating isolates of E. faecalis to the subspecies level in addition to identifying outbreak isolates.

Base Sequence↗

PubMed2000

Pharmacodynamics of vancomycin and ampicillin alone and in combination with gentamicin once daily or thrice daily against Enterococcus faecalis in an in vitro infection model.

We compared the pharmacodynamic activities of vancomycin and ampicillin with or without gentamicin once daily or thrice daily in an in vitro infection model with fibrin-platelet clots (FPCs) infected with Enterococcus faecalis. Antibiotics were administered as a bolus to simulate human pharmacokinetics with regimens consisting of vancomycin 1 g q12h, ampicillin 2 g q6h and gentamicin 1.3 mg/kg q8h and 5 mg/kg qd. Model experiments were performed in duplicate over 72 h. FPCs were removed from the models in triplicate at 0, 8, 24, 32, 48 and 72 h, weighed, homogenized, diluted and plated to determine colony counts. Additional FPCs were removed at over 72 h post-antibiotic dose to determine antibiotic concentrations. The inoculum density at 72 h was used to compare bactericidal activity between the regimens. Overall, all antibiotic regimens containing either ampicillin or vancomycin significantly (P < 0.01) decreased the bacterial load at 72 h compared with the growth control although monotherapy regimens with either vancomycin or gentamicin had little impact. Ampicillin was superior to vancomycin with or without the addition of gentamicin (P < 0.01). There were no significant differences in reduction of bacterial density at 72 h between the combination of ampicillin or vancomycin plus gentamicin q8h and ampicillin or vancomycin plus gentamicin once daily. This was despite achieving unmeasurable FPC gentamicin concentrations after the 8 h time point during the once-daily aminoglycoside regimen. Vancomycin plus gentamicin either every 8 h or once daily was significantly (P < 0.01) better than vancomycin alone. Ampicillin plus either of the two gentamicin regimens was also better than ampicillin alone but this did not reach statistical significance. Our data suggest that once-daily gentamicin in combination with ampicillin or vancomycin demonstrates equivalent bacterial reductions to combination therapy with thrice-daily gentamicin. Once-daily aminoglycoside combination therapy for the treatment of enterococcal endocarditis warrants further investigation.

Ampicillin↗

PubMed1991

Vancomycin-resistant Enterococcus faecalis in a bone-marrow transplant recipient.

Since 1988 a number of reports on the emergence of vancomycin-resistant enterococci have been published. We describe an additional case of colonization of and subsequent infection with a vancomycin-resistant Enterococcus faecalis in a bone-marrow transplant recipient, who had never before received vancomycin therapy. The strain was resistant to most antibiotics tested, including low-level resistance to gentamicin and cross-resistance to teicoplanin. It was sensitive to amoxicillin and the lipopeptide antibiotic daptomycin. The origin, the mode of acquisition and the incidence of vancomycin-resistant enterococci are still unknown. The clinical and bacteriological features of the cases reported thus far and the genetic and biochemical basis of vancomycin resistance are discussed.

Adult↗

PubMed1999

Influence of adjunctive interferon-gamma on treatment of gentamicin- and vancomycin-resistant Enterococcus faecalis infection in mice.

Increasing antibiotic resistance and the development of multidrug-resistance in the enterococci has complicated the treatment of serious enterococcal infections. It has been demonstrated in vitro that interferon-gamma (IFN-gamma) significantly augments the activities of gentamicin and vancomycin against Enterococcus faecalis resistant to these antibiotics. The present study was aimed at determining whether this beneficial effect of IFN-gamma on antienterococcal antibiotic activity can be validated in vivo. Following intraperitoneal inoculation in mice with a gentamicin- and vancomycin-resistant E. faecalis clinical isolate, the animals received IFN-gamma, antibiotic or a combination of both agents, subcutaneously, at determined dosing regimens. Treatment with IFN-gamma alone significantly improved survival of infected animals in a dose-dependent manner. High dose IFN-gamma was not beneficial and the level of enterococcal infectious burden influenced the effectiveness of the cytokine. The addition of IFN-gamma to therapy with gentamicin or vancomycin, or a combination of both antibiotics was associated with a marked increase in survival of infected non-neutropenic mice compared to treatments with the agents alone. However, the same treatments made in infected neutropenic mice did not show an enhancement effect by IFN-gamma after a combination therapy with antibiotics. In a study to examine pharmacokinetic interactions, concurrent administration with IFN-gamma significantly modified the disposition of gentamicin but not that of vancomycin. The results of this study suggest that the use of IFN-gamma in combination with vancomycin or gentamicin is a new treatment option that might improve the outcome of therapy of multidrug-resistant E. faecalis infections.

Animals↗

PubMedDate not supplied

Bactericidal activity of gatifloxacin (AM-1155) against Pseudomonas aeruginosa and Enterococcus faecalis in an in vitro bladder model simulating human urinary concentrations after oral administration.

The bactericidal activity of gatifloxacin, a new 6-fluoro-8-methoxy quinolone, was determined in a dynamic in vitro model mimicking complicated lower urinary tract infection. Strains of Pseudomonas aeruginosa and Enterococcus faecalis with different susceptibility were exposed to changing gatifloxacin concentrations, simulating human urinary concentrations afer oral treatment with 200 mg twice daily for 3 consecutive days. Bacterial numbers of P. aeruginosa (minimal inhibitory concentrations, MIC: < or =32 microg/ml) and of E. faecalis (MIC: 16 microg/ml) were reduced to undetectable levels during exposure. For the strains with lower susceptibility, gatifloxacin showed bactericidal activity, but eradication was not complete. Thus, in a complicated urinary tract infection model, breakpoint MICs of gatifloxacin for uropathogenic organisms were presumed to range from 16 to 32 microg/ml. At least 86% of recent clinical isolates of P. aeruginosa and E. faecalis were inhibited at its breakpoint MIC. These results suggest that gatifloxacin may be useful in the treatment of urinary tract infections.

Administration, Oral↗

PubMed1991

Localization of translocating Escherichia coli, Proteus mirabilis, and Enterococcus faecalis within cecal and colonic tissues of monoassociated mice.

Normal bacteria can translocate (migrate) across an intact intestinal mucosa, but the anatomical site of bacterial translocation has not been defined. Gastrointestinal (GI) cross-sections were obtained from mice monoassociated with high cecal concentrations of Escherichia coli, Proteus mirabilis, or Enterococcus faecalis. As previously reported (C. L. Wells, R. P. Jechorek, and K. J. Gillingham, Arch. Surg. 126:247-252, 1991), these mice had viable translocating bacteria recovered from mesenteric lymph nodes and livers. No abnormal GI histology was noted. Immunofluorescence was used to localize GI bacteria, and similar observations were made with each of the three bacterial species. Smaller numbers (P less than 0.01) of bacteria were observed in stomach and small intestinal tissues than in cecal and colonic tissues, suggesting that the preferred site of tissue penetration for intestinal E. coli, P. mirabilis, and E. faecalis might be the cecum or colon as opposed to the stomach or small intestine.

Animals↗

PubMed1997

Regulation of transfer of the Enterococcus faecalis pheromone-responding plasmid pAD1: temperature-sensitive transfer mutants and identification of a new regulatory determinant, traD.

The enterococcal, conjugative, cytolysin plasmid pAD1 confers a mating response to the peptide sex pheromone cAD1 secreted by plasmid-free strains of Enterococcus faecalis. Cells carrying pAM714, a pAD1::Tn917 derivative with wild-type conjugation properties, were mutagenized with ethyl methanesulfonate to obtain variants that were induced (in the absence of pheromone) to transfer plasmid DNA upon shifting from 32 to 42 degrees C. Of 31 such mutants generated, the results of analyses of 7 are presented in detail. All seven strains were thermosensitive in the E. faecalis host FA2-2; colony morphology, clumping, and DNA transfer correlated well with each other at the two temperatures. In the nonisogenic host E. faecalis OG1X, however, only one derivative (pAM2725) exhibited correlation of all three traits at both temperatures. Three (pAM2700, pAM2703, and pAM2717) clumped and had colonies characteristic of pheromone-induced cells at 32 degrees C but transferred plasmid DNA at a higher frequency only at the elevated temperature. The other three (pAM2708, pAM2709, and pAM2712) were derepressed at both temperatures for all three characteristics. Four of the mutations, including that of pAM2725, mapped within the traA determinant, whereas two mapped identically in a previously unnoted open reading frame (designated traD) putatively encoding a short (23-amino-acid) peptide downstream of the inhibitor peptide determinant iad and in the opposite orientation. One mutant could not be located in the regions sequenced. Studies showed that the traA and traD mutations could be complemented in trans with a DNA fragment carrying the corresponding regions.

Amino Acid Sequence↗