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At least 19 recordsLinked to original sources
PubMed1992

Identical genes confer high-level resistance to gentamicin upon Enterococcus faecalis, Enterococcus faecium, and Streptococcus agalactiae.

The structural gene coding for the bifunctional aminoglycoside-modifying 6'-acetyltransferase-2''-phosphotransferase (6'AAC-2''APH) enzyme was specifically amplified by the polymerase chain reaction using template DNA of clinical isolates of enterococci (both Enterococcus faecalis and Enterococcus faecium) and the single high-level gentamicin-resistant Streptococcus agalactiae strain identified thus far. The results of the present study demonstrated that the genes encoding this antibiotic resistance trait are highly homologous in these species. In dot blot hybridization assays using nonradioactively labeled oligonucleotide probes, strains with and without high-level gentamicin resistance could be discerned unequivocally. The gene segment encoding 6'-acetylating activity and the gene segment encoding 2''-phosphorylating activity were simultaneously present in all isolates exhibiting high-level gentamicin resistance.

Acetyltransferases↗
PubMed2001

Distribution of aminoglycoside resistance genes in recent clinical isolates of Enterococcus faecalis, Enterococcus faecium and Enterococcus avium.

Aminoglycoside modifying enzymes (AMEs) are major factors which confer aminoglycoside resistance on bacteria. Distribution of genes encoding seven AMEs was investigated by multiplex PCR for 279 recent clinical isolates of enterococci derived from a university hospital in Japan. The aac(6')-aph(2"), which is related to high level gentamicin resistance, was detected at higher frequency in Enterococcus faecalis (42.5%) than in Enterococcus faecium (4.3%). Almost half of E. faecalis and E. faecium isolates possessed ant(6)-Ia and aph(3')-IIIa. The profile of AME gene(s) detected most frequently in individual strains of E. faecalis was aac(6')aph(2") + ant(6)-Ia + aph(3')-IIIa, and isolates with this profile showed high level resistance to both gentamicin and streptomycin. In contrast, AME gene profiles of aac(6')-Ii+ ant(6)-Ia+aph(3')-IIIa, followed by aac(6')-Ii alone, were predominant in E. faecium. Only one AME gene profile of ant(6)-Ia+aph(3')-IIIa was found in Enterococcus avium. The ant(4')-Ia and ant(9)-Ia, which have been known to be distributed mostly among Staphylococcus aureus strains, were detected in a few enterococcal strains. An AME gene aph(2")-Ic was not detected in any isolates of the three enterococcal species. These findings indicated a variety of distribution profiles of AME genes among enterococci in our study site.

Aminoglycosides↗
PubMed2002

Characterization of anti-Listeria monocytogenes bacteriocins from Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis strains isolated from Raïb, a Moroccan traditional fermented milk.

Seventy-four samples of raïb, a Moroccan traditional fermented milk, were screened for their anti-Listeria monocytogenes activity. Nine lactic acid bacteria with antilisterial activity were isolated and identified as Lactococcus lactis [4], Enterococcus faecium [4], and E. faecalis [1]. Antibacterial spectra, determined against 45 target strains, led to the selection of four antibacterial-producing strains, which were further characterized. Their anti-microbial agents, inactivated by one or more proteases, were designed as bacteriocins. Lactococcin R9/2 and R10/1 showed the broadest range of inhibitory action. Anti-bacterial spectra and physico-chemical properties suggest that these bacteriocins were similar to nisin. Enterocin R69 had a specificity of action against Listeria spp., whereas Enterocin R18 had a broad spectrum of activity. Lc. lactis R9/2 and E. faecalis R18 were able to coagulate sterilised UHT milk at 30 degrees C in 24 h and induced a 2 log reduction in L. monocytogenes ATCC 15313 population.

Animals↗
PubMed2000

Virulence factors of Enterococcus faecalis and Enterococcus faecium blood culture isolates.

Known and potential virulence factors of enterococcal blood culture isolates were studied using 89 Enterococcus faecalis and 24 Enterococcus faecium isolates. The prevalence of the respective factors was (Enterococcus faecalis vs. Enterococcus faecium): hemolysin 16% vs. 0%, gelatinase 55% vs. 0%, aggregation substance 63% vs. 13%, lipase 35% vs. 4%, hemagglutinin 97% vs. 0%. Deoxyribonuclease was not detected in any isolate. The study showed that hemagglutinin and lipase may represent additional virulence factors of Enterococcus faecalis but not Enterococcus faecium. The significance of these factors in the pathogenesis of enterococcal infection needs to be elucidated in further studies.

Bacterial Proteins↗
PubMed2003

Analysis of daptomycin efficacy and breakpoint standards in a murine model of Enterococcus faecalis and Enterococcus faecium renal infection.

Daptomycin efficacy against clinical isolates of Enterococcus faecalis, Enterococcus faecium, and a lab-derived daptomycin-resistant isolate of E. faecalis was investigated in a mouse model of renal infection. The daptomycin MICs against these enterococci ranged from 0.5 to 50 micro g/ml. The objective of this study was to determine the relationship between the MICs of drugs against E. faecalis and E. faecium and the level of daptomycin exposure needed to evaluate the drug's efficacy. Correlating the required therapeutic exposures of mice with the exposures achieved clinically allowed us to project enterococcal breakpoint values. Mice pretreated with carrageenan were infected intravenously with 3 x 10(8) to 4 x 10(8) CFU of E. faecalis or E. faecium. Daptomycin (5 to 50 mg of drug/kg of body weight) or saline control was administered 4 h postinfection and continued once daily for 2 days (three total doses). On day 4, infected kidneys were harvested, homogenized, and dilution plated. Efficacy was defined as a > or = 2-log(10) (99%) reduction in bacterial burden in infected kidneys. At clinically relevant dosages and exposures (area under the curve, 400 to 600 microg.hr/ml), daptomycin demonstrated similar and marked efficacy against all clinical enterococcal isolates tested. Daptomycin achieved efficacy with comparable doses against both vancomycin-sensitive (MIC, < or = 4 microg/ml) and -resistant enterococcal strains tested. Efficacy was also established against the lab-derived daptomycin-resistant E. faecalis isolate. In this murine renal infection model, clinically relevant exposures of daptomycin were effective against E. faecalis and E. faecium strains for which MICs were < or = 8 microg/ml. These murine efficacy data for daptomycin, along with surveillance data and human pharmacokinetic exposures achieved, suggest a breakpoint concentration value of < or = 8 microg/ml (susceptible) and > or = 16 microg/ml (resistant) for daptomycin against E. faecium and E. faecalis.

Animals↗
PubMed1998

In vitro activity of quinupristin/dalfopristin and newer quinolones combined with gentamicin against resistant isolates of Enterococcus faecalis and Enterococcus faecium.

In a study designed to obtain data on compounds active against enterococci, the minimum inhibitory concentrations (MICs) of quinupristin/dalfopristin (RP 59500) and the novel quinolones DU-6859a, trovafloxacin, levofloxacin, and sparfloxacin were determined for 122 Enterococcus faecalis and seven Enterococcus faecium isolates. In addition, 15 Enterococcus faecalis isolates resistant to gentamicin, DU-6859a, and trovafloxacin were exposed over time to combinations of DU-6859a plus gentamicin and trovafloxacin plus gentamicin. DU-6859a and trovafloxacin were found to be the most active compounds against Enterococcus faecalis and DU-6859a and RP 59500 against Enterococcus faecium. Synergy between either DU-6859a or trovafloxacin and gentamicin was observed with 27 to 35% of the isolates. It is concluded that DU-6859a and trovafloxacin are very potent against enterococci, especially when combined with gentamicin.

Anti-Bacterial Agents↗
PubMed2004

VanD-type vancomycin-resistant Enterococcus faecium and Enterococcus faecalis.

Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Amino Acid Sequence↗
PubMed2005

PCR amplification of a species-specific putative transcriptional regulator gene reveals the identity of Enterococcus faecalis.

Enterococcus faecalis, an opportunistic bacterial pathogen, has become prominent in recent decades due to its ability to develop resistance to various antibiotics. The ability to rapidly identify E. faecalis, followed by prompt treatment, is therefore vital for the control of this pathogen. In this report, upon comparative BLAST search of the E. faecalis genome against other bacterial DNA sequences at GenBank, an E. faecalis-specific gene Ef0027, that codes for a putative phosphosugar binding transcriptional regulator, was identified. Using PCR primers derived from this gene (i.e., Ef0027F/R), a specific DNA fragment of 518 bp was detected in E. faecalis strains only, and it was not found in other Enterococcus species and common bacteria. These results suggest that PCR amplification of the putative transcriptional regulator gene Ef0027 provides a useful means of identifying E. faecalis from other bacteria.

Computational Biology↗
PubMed1993

Evaluation of modified MicroScan screening tests for high-level aminoglycoside resistance in Enterococcus faecalis.

Enterococcus faecalis strains that are refractory to aminoglycoside-penicillin synergistic killing can be predicted by their ability to grow in high concentrations of aminoglycoside (2,000 mg/L) or by disk diffusion tests using high content aminoglycoside disks. Forty-eight well-characterized clinical isolates of E. faecalis were used to evaluate recently reformulated gentamicin and streptomycin synergy screening tests on both overnight and rapid MicroScan (MS) Pos MIC panels (Baxter, West Sacramento, CA). The results obtained with the MS screening tests were compared with those from modified agar disk diffusion results. Discrepancies were resolved by an in-house broth microdilution method. The MS overnight tests detected 25 of 25 (100%) gentamicin high-level resistant strains and 23 of 24 (96%) streptomycin high-level resistant strains. No false high level resistance was found with the MS overnight tests. The MS rapid tests detected 100% of the gentamicin and 100% of the streptomycin high-level resistant strains, but one isolate was falsely high-level resistant to gentamicin (96% specific). The reformulated MS synergy screening tests were acceptable alternatives to disk diffusion for detection of high level aminoglycoside resistance in E. faecalis.

Anti-Bacterial Agents↗
PubMed1988

Effects of medium and inoculum variations on screening for high-level aminoglycoside resistance in Enterococcus faecalis.

Enterococcus faecalis isolates that are refractory to aminoglycoside-penicillin synergy can be detected by their ability to grow in the presence of high concentrations of aminoglycoside (2,000 micrograms/ml). In past studies investigators have used a variety of media and inoculum sizes to perform high-level aminoglycoside resistance screens, but little is known about how these variations affect test accuracy. We screened 63 E. faecalis strains on different media by using various inoculum sizes and correlated the results with synergy test results obtained by time-kill studies. Screens were done with dextrose-phosphate agar, brain heart infusion agar, Trypticase soy agar with 5% sheep blood, Mueller-Hinton agar with 5% sheep blood, dextrose-phosphate broth, and Mueller-Hinton broth. Agar screens were inoculated with 10(2), 10(4), and 10(6) CFU; and broth screens contained a final inoculum of 10(5) CFU/ml. The E. faecalis isolates were tested for high-level resistance to streptomycin, kanamycin, amikacin, gentamicin, and tobramycin. Of the 63 isolates tested, 21 did not show high-level resistance to any of the aminoglycosides tested, and 42 demonstrated high-level resistance to one or more drugs. The sensitivity of most screens was greater than or equal to 90%. Regardless of the inoculum size or medium used, false-resistance results were seldom encountered. Screen specificity, which was used as the indicator of false susceptibility, was markedly influenced by both the inoculum size and the drug being tested. Specificity was low whenever a 10(2)-CFU inoculum was used, when amikacin was tested with any inoculum, and when tobramycin was tested in broth media. Data for kanamycin could be used to predict amikacin-penicillin synergy, and the highly accurate gentamicin screen obviated the need for the testing of tobramycin. We recommend a 10(6) -CFU inoculum for agar screens and a 10(5) -CFU/ml inoculum for broth screens. The type of medium used did not substantially influence screen accuracy. Among the aminoglycosides, only streptomycin, gentamicin, and occasionally, kanamycin need to be used to screen E. faecalis isolates for aminoglycoside-penicillin synergy.

Amikacin↗
PubMed2006

Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis.

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract. E. faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E. faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E. faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E. faecalis mutant strains carrying defined mutations in several genes, including ccfA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E. faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and delta eep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E. faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens.

Conjugation, Genetic↗
PubMed2002

Pulmonary hypertension syndrome in young chickens challenged with frozen and autoclaved cultures of Enterococcus faecalis.

Enterococcus faecalis, when administered in a growth medium or sterile saline, will cause pulmonary hypertension syndrome (PHS) in chickens. The objective of this study was to determine if frozen and/or autoclaved cultures of E. faecalis retain ability to evoke PHS. In Trial 1, chicks were inoculated with 3.6 x 10(7) E. faecalis (IA) in tryptic soy broth (TSB) from either a live culture or one that had been autoclaved (120 degrees C for 20 min). Controls received TSB. Autoclaved and live cultures produced the same degree of PHS in a majority of the birds. Trial 2 used the same protocol, except a frozen (-70 degrees C for 60 min) culture of E. faecalis was compared with the control. The results agreed with those of Trial 1, i.e., the frozen culture also produced PHS. Trial 3 was conducted to determine if E. faecalis caused PHS by producing and releasing some unknown substance into the supernatant. Incidence of PHS was based on percentage of birds exhibiting ascites fluid at 24 hr after challenge. Controls received sterile, frozen, or autoclaved TSB. As compared with controls, those birds that received challenge with E. faecalis alone, supernatant alone, and E. faecalis plus supernatant from live cultures exhibited similar incidence of ascites, whereas birds that received E. faecalis plus supernatant and supernatant alone from cultures that had been either frozen or autoclaved exhibited elevated incidence of ascites as compared with controls. Also, with frozen and autoclaved cultures, those birds that received only pelleted E. faecalis exhibited incidence of ascites that did not differ from controls. Apparently, E. faecalis produces PHS in chicks by producing and releasing an unknown toxin.

Animals↗
PubMed1991

Cloning and nucleotide base sequence analysis of a spectinomycin adenyltransferase AAD(9) determinant from Enterococcus faecalis.

Enterococcus faecalis LDR55, a human clinical isolate, is resistant to tetracycline (Tcr), erythromycin (Emr), and high levels (greater than 2,000 micrograms/ml) of spectinomycin (Spr) but not streptomycin. Filter matings between strain LDR55 and E. faecalis OG1-RF produced transconjugants with the following resistance phenotypes: Tcr Emr Spr, Tcr Emr, Tcr Spr, and Tcr only but never Emr or Spr only. The genetic determinant encoding resistance to spectinomycin was cloned in Streptococcus sanguis Challis from pDL55, a 26-kb plasmid harbored by a Tcr Spr transconjugant. Subcloning experiments yielded a 1.1-kb ClaI-NdeI fragment that encoded very high-level Spr in S. sanguis (10 mg/ml) and Escherichia coli (50 mg/ml). Cell extracts of cultures obtained from Spr strains expressed adenylating activity for spectinomycin but not for streptomycin, indicating that Spr was due to an AAD(9) activity. The nucleotide base sequence of the 1.1-kb ClaI-NdeI fragment contained a single 750-base open reading frame. The protein predicted from the open reading frame consisted of 250 amino acids and had a calculated size of approximately 28,000 daltons, similar to the size estimated from maxicell analysis (29,000 daltons). The deduced amino acid sequence of the streptococcal AAD(9) was compared with that of the AAD(9) encoded by staphylococcal transposon Tn554. The two proteins shared approximately 39% amino acid identity, which was expanded to 53% when conservative amino acid changes were included. When the streptococcal protein was compared with an AAD(3")(9) protein of E. coli, the degrees of identity were 27 and 47%, on the basis of actual amino acids and conservative replacements, respectively. The cloning and nucleotide base sequence analyses of the spectinomycin AAD(9) determinant from E. faecalis that results in high-level Spr when transferred to S. sanguis or E. coli are presented.

Base Sequence↗
PubMed2004

Esp-independent biofilm formation by Enterococcus faecalis.

Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of E. faecalis is critical for biofilm formation by this organism. However, in the same study, esp-deficient strains of E. faecalis were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by E. faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence. Using scanning electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E. faecalis. In summary, E. faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.

Bacterial Proteins↗
PubMed1990

A transferable plasmid associated with AS-48 production in Enterococcus faecalis.

Enterococcus faecalis S-48 produces a peptide antibiotic, AS-48, and a bacteriocin, Bc-48. We have isolated mutants that lack these inhibitory characteristics. Further analysis of the mutants indicates that a plasmid of 56 kilobases (pMB2) may harbor the genes for AS-48. In conjugation experiments, pMB2 has been transferred into a plasmid-free OG1X strain of E. faecalis. The OG1X(pMB2) transconjugant produces the antibiotic AS-48 in solid medium, and the MIC of AS-48 for this strain is the same as that of the donor strain.

Anti-Bacterial Agents↗
PubMed2004

Effects of iron and phytic acid on production of extracellular radicals by Enterococcus faecalis.

Enterococcus faecalis is a human intestinal commensal that produces extracellular superoxide, hydrogen peroxide, and hydroxyl radical while colonizing the intestinal tract. To determine whether dietary factors implicated in colorectal cancer affect oxidant production by E. faecalis, radicals were measured in rats colonized with this microorganism while on diets supplemented with iron or phytic acid. Hydroxyl radical activity was measured by assaying for aromatic hydroxylation products of D-phenylalanine using reverse-phase high-performance liquid chromatography and electrochemical detection. In vitro, as expected, iron enhanced, and phytic acid decreased, hydroxyl radical formation by E. faecalis. For rats colonized with E. faecalis given supplemental dietary iron (740 mg elemental iron as ferric phosphate per kg diet) or phytic acid (1.2% w/w), no differences were found in concentrations of urinary ortho- or meta- isomers of D-phenylalanine compared to rats on a basal diet. Aqueous radicals in colonic contents were further assessed ex vivo by electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide as a spin trap. Mixtures of thiyl (sulfur-centered) and oxygen-centered radicals were detected across all diets. In vitro, similar spectra were observed when E. faecalis was incubated with hydrogen sulfide, air-oxidized cysteine, or an alkylsulfide, as typical sulfur-containing compounds that might occur in colonic contents. In conclusion, intestinal colonization with E. faecalis in a rat model generates both thiyl and oxygen-centered radicals in colonic contents. Radical formation, however, was not significantly altered by short-term dietary supplementation with iron or phytic acid.

Animals↗
PubMed1995

[Meningitis due to Enterococcus faecalis].

Enterococcus faecalis meningitis is an infrequent entity that accounts for less than 1% of all suppurative meningitis in the adult. Usually, this infection affects patients with compromised host defenses or those who have congenital or acquired CNS lesions mainly as intrahospitalary infections. An 85 year old woman from our community (Tandil county) without any predisposed condition, was admitted in the hospital in an unconscious state (grade 3 Glasgow's index), meningeal signs and purulent CSF, from which E. faecalis was isolated. The patient was treated with IV Ampicillin and Gentamycin (17 days), intrathecal Gentamycin (4 days) and IV dexametasona (6 days). The clinical and bacteriological remission was achieved, without any sequel or relapse during 2 years follow up.

Aged↗
PubMed2004

The three-dimensional structure of catalase from Enterococcus faecalis.

Enterococcus faecalis haem catalase was crystallized using lithium sulfate at neutral pH. The crystals belong to space group R3, with unit-cell parameters a = b = 236.9, c = 198.1 A. The three-dimensional structure was determined by molecular replacement using a subunit of the Proteus mirabilis catalase structure. It was refined against 2.3 A synchrotron data to a free R factor of 21.8%. Like other catalases, the E. faecalis catalase is a homotetramer with a fold and structure similar to those of its structurally closest relative P. mirabilis. The solvent structure in the active site is identical in the four subunits but differs from that found in other catalases. The structural consequences of the Ramachandran outlier Ser196 are discussed.

Amino Acid Sequence↗