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PubMed1992

Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus.

Conjugative transfer, in the apparent absence of plasmid DNA, of high-level vancomycin resistance from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus B111 has been demonstrated in vivo and in vitro. Selection of transconjugants on media containing erythromycin or chloramphenicol may result in the transfer of resistance to erythromycin, chloramphenicol, gentamicin, streptomycin and vancomycin though these are capable of separate transfer. Vancomycin resistance has not been transmitted from staphylococcus to staphylococcus though transfer of erythromycin and of chloramphenicol resistance has been achieved.

Conjugation, Genetic↗
PubMed1992

The putative Na+/H+ antiporter (NapA) of Enterococcus hirae is homologous to the putative K+/H+ antiporter (KefC) of Escherichia coli.

Two monovalent ion porters, the putative Na+/H+ antiporter (NapA) of Enterococcus hirae and the putative K+/H+ antiporter (KefC) of Escherichia coli, are similar in sequence throughout their hydrophobic domains. These two proteins, which comprise a novel family of transporters unrelated to the previously characterized Na+/H+ exchangers of E. coli (NhaA and NhaB) are proposed to function by essentially the same mechanism.

Amino Acid Sequence↗
PubMed1996

Rapid identification of Streptococcus and Enterococcus species using diffuse reflectance-absorbance Fourier transform infrared spectroscopy and artificial neural networks.

Diffuse reflectance-absorbance Fourier transform infrared spectroscopy (FT-IR) was used to analyse 19 hospital isolates which had been identified by conventional means to one Enterococcus faecalis, E. faecium, Streptococcus bovis, S. mitis, S. pneumoniae, or S. pyogenes. Principal components analysis of the FT-IR spectra showed that this 'unsupervised' learning method failed to form six separable clusters (one of each species) and thus could not be used to identify these bacteria base on their FT-IR spectra. By contrast, artificial neural networks (ANNs) could be trained by 'supervised' learning (using the back-propagation algorithm) with the principal components scores of derivatised spectra to recognise the strains from their FT-IR spectra. These results demonstrate that the combination of FT-IR and ANNs provides a rapid, novel and accurate bacterial identification technique.

Algorithms↗
PubMed1992

Isolation and characterization of genetic expression and secretion signals from Enterococcus faecalis through the use of broad-host-range alpha-amylase probe vectors.

We have constructed two broad-host-range Gram+/Gram- probe vectors designed for the cloning of bacterial genetic expression and secretion signals. These vectors make use of a silent reporter gene encoding the mature alpha-amylase from Bacillus licheniformis whose reactivation can easily be monitored on iodine-stained starch plates. Shotgun cloning of Enterococcus faecalis DNA fragments allowed recovery of several cassettes directing transcription, translation of the reporter gene and secretion of alpha-amylase. Sequence analysis revealed, in each case, the presence of a putative promoter, ribosome-binding site and signal peptide similar to those described in other Gram+ bacteria.

Amino Acid Sequence↗
PubMed1992

A strategy to construct vector-free amylolytic strains through nondisruptive homologous recombination: application to Enterococcus faecalis.

In order to direct chromosomal integration of the alpha-amylase-encoding gene from Bacillus licheniformis (amyL) under the control of expression and secretion signals from Enterococcus faecalis, the chromosomal fragment (named AB) from the pGIP3124 plasmid [Hols et al., Gene 118 (1992) 21-30] was chosen and split into two fragments (A and B). A translation fusion between the A fragment and 'amyL, deleted of its expression and secretion signals, was made and this fusion was flanked with the AB fragment at its right end. The A::'amyL:AB integration module was cloned into a thermosensitive pE194 replicon (chloramphenicol resistant; CmR) and electro-transformed into E. faecalis OG1X. After an overnight culture in selective liquid medium, the offspring from the amylolytic transformants obtained was shown to yield CmR colonies with two distinct halo sizes on iodine-stained starch plates. Southern analysis clearly showed that the smaller halos corresponded to descendants in which the plasmid had integrated into the chromosome through homologous recombination. One such Amy+ integrant in the AB site was further cultured under nonselective conditions at 42 degrees C for about 20 generations, and the offspring was screened for Amy+/CmS clones. Such revertants were indeed found, and Southern analysis clearly showed that the vector matrix had been excised through homologous recombination between the redundant A sites, leaving the integrated amy gene intact.

Amino Acid Sequence↗
PubMed1992

Sequence of the vanC gene of Enterococcus gallinarum BM4174 encoding a D-alanine:D-alanine ligase-related protein necessary for vancomycin resistance.

The amplification product obtained with DNA from vancomycin-resistant (VmR) Enterococcus gallinarum BM4174 and a pair of degenerate oligodeoxyribonucleotides that correspond to conserved amino acid (aa) motifs in Escherichia coli D-alanine (D-Ala):D-Ala ligases and in En. faecium VmR protein (VanA) was used as a probe to clone the vanC gene of that strain. The vanC product, with a calculated Mr of 37,504, exhibits 29 to 38% aa identity with VanA and E. coli ligases. Insertional inactivation of vanC led to Vm sensitivity of BM4174 suggesting that the gene may encode a D-Ala:D-Ala ligase of altered specificity.

Amino Acid Sequence↗
PubMed1995

Characterization and heterologous expression of the tetL gene and identification of iso-ISS1 elements from Enterococcus faecalis plasmid pJH1.

The tetracycline-resistance (TcR) determinant of the Enterococcus faecalis plasmid pJH1 has been identified and located on a 2.2-kb RsaI-EcoRI fragment. The fragment was cloned in Escherichia coli, and specified TcR in this host. The nucleotide (nt) sequence of the cloned fragment showed the presence of an open reading frame (ORF) of 1374 bp, designated tetL. The nt sequence of tetL from pJH1 was identical to that of the tetL present on pLS1 from Streptococcus agalactiae. Upstream of the pJH1 tetL, part of another ORF was found that, except for two single-nt substitutions, was identical to an iso-ISS1 element from Lactococcus lactis. Hybridization studies indicated the presence of several ISS1-like elements in plasmid pJH1, but not on the En. faecalis chromosome. To study its usefulness as a marker in Gram+ organisms, the pJH1 tetL was cloned on the broad-host-range plasmid pNZ124, resulting in pNZ280, that was found to give resistance to 40 micrograms Tc/ml in Lc. lactis and Bacillus subtilis.

Amino Acid Sequence↗
PubMed1996

Mobilization of vancomycin resistance by transposon-mediated fusion of a VanA plasmid with an Enterococcus faecium sex pheromone-response plasmid.

A striking feature of recent outbreaks of vancomycin-resistant (VmR) enterococci is the apparent horizontal dissemination of resistance determinants. The plasmids pHKK702 and pHKK703 from Enterococcus faecium clinical isolate R7 have been implicated in the conjugal transfer of VmR. pHKK702 is a 41-kb plasmid that contains an element indistinguishable from the glycopeptide-resistance transposon Tn1546. pHKK703 is an approx. 55-kb putative sex pheromone-response plasmid that is required for conjugative mobilization of pHKK702. During experiments in which strain R7 was used as a donor, a highly conjugative VmR transconjugant was isolated that formed constitutive cellular aggregates. Restriction analyses and DNA hybridizations revealed that the transconjugant harbored a single plasmid of approx. 92 kb and this plasmid (pHKK701) was composed of DNA from both pHKK702 and pHKK703. Results from DNA sequence analyses showed that a 39-kb composite transposon (Tn5506) from pHKK702 had inserted into pHKK703. The left end of Tn5506 contained a single insertion sequence (IS) element, IS1216V2, whereas the right end was composed of a tandem IS structure consisting of the novel 1065-bp IS1252 nested within an IS1216V1 element. Transposition of Tn5506 from pHKK702 to pHKK703 created an 8-bp target sequence duplication at the site of insertion and interrupted an ORF (ORFX) that was 91% identical to that of prgX, a gene proposed to negatively regulate sex pheromone response of the E.faecalis plasmid, pCF10. We propose that the interruption of ORFX by Tn5506 led to the constitutive cellular aggregation phenotype and thereby enhanced the efficiency with which VmR was transferred. Similar IS1216V-mediated transposition events may contribute to the horizontal spread of glycopeptide resistance among enterococci in nature.

Amino Acid Sequence↗
PubMed1996

Characterization of Tn1547, a composite transposon flanked by the IS16 and IS256-like elements, that confers vancomycin resistance in Enterococcus faecalis BM4281.

A 64-kb genetic element harboring a vanB vancomycin-resistance (VmR) gene cluster was shown to translocate from the chromosome of Enterococcus faecalis BM4281 into the hemolysin (Hly) plasmid, pIP964. Sequence analysis of the resulting junction fragments indicated that the VmR genes were carried by a composite transposon, Tn1547, bounded by two distantly related insertion sequences (IS), designated IS256-like and IS16, in a direct orientation. IS256-like (1324 bp) was identical to IS256, except for two nucleotide (nt) transitions in the putative transposase gene. IS16 (1466 bp) contained a large open reading frame (ORF) that was 61% identical to the gene encoding the putative transposase of IS256. There was 58% identity between the deduced amino acid (aa) sequences of the putative transposases of IS256-like (390 aa) and IS16 (395 aa). IS16 was delineated by imperfect inverted repeats (IR) (18 out of 26 bp) which were related (23/26 bp identity) to their respective imperfect IR counterparts in IS256. The difference between the IS was not a barrier for transposition of Tn1547 which generated an 8-bp duplication at the target site. Dissemination of VanB-type resistance among enterococci results from two mechanisms: (i) large conjugative elements translocate from chromosome to chromosome following inter-strain transfer; and (ii) as described in this report, the VmR genes transpose from replicon to replicon within the same strain as part of composite transposons that are internal to the conjugative elements.

Amino Acid Sequence↗
PubMed1996

The humoral immune response of turbot to recently isolated pathogenic Enterococcus strains. Cross-reactivity with other Gram-positive bacteria.

An Enterococcus sp. causing severe mortalities among farmed turbot (Scophthalmus maximus) has recently been detected in northwest Spain. We found that specific turbot serum antibodies raised against isolate RA-99.1 of the new pathogen by intraperitoneal immunization, did not cross react in enzyme-linked immunosorbent assay (ELISA) with other enterococcal or non-enterococcal Gram-positive bacteria. In immunoblotting, antibodies raised against strain RA-99.1 recognized the same components in the homologous total soluble antigen preparation (TSA) as in TSA of two other isolates of the pathogen obtained from different farms. Anti-RA-99.1 serum also recognized some components of the TSA of several other Gram-positive bacteria. Immunogold labelling indicated that the antigens which provoke the humoral immune response to this pathogen are located mainly on the bacterial surface.

Animals↗
PubMedDate not supplied

Effect of blood product medium supplements on the activity of cefotaxime and other cephalosporins against Enterococcus faecalis.

The activities of cefotaxime and other aminothiazoyl oxime cephalosporins against Enterococcus faecalis were enhanced by addition of 5% sheep blood to Mueller-Hinton agar. This effect was not seen with aztreonam (aminothiazoyl oxime monobactam), cefotiam (aminothiazoyl, nonoxime), or other cephalosporins, and it was specific to the syn-configuration of the oxime moiety. Enhancement of cefotaxime activity was demonstrable against approximately 50% of 86 clinical isolates and could only be shown at low bacterial inocula. Human serum, serum alpha 1-, beta- and gamma-globulin fractions and albumin often antagonized or did not affect significantly the antimicrobial activity of cefotaxime, while the alpha 2-globulin fraction usually enhanced drug activity. The in vivo activity of cefotaxime against E. faecalis was examined in a rat peritoneal abscess model. The test organism was resistant to cefotaxime by standard methods (MIC greater than 128 micrograms/ml) but was inhibited by 1.0 microgram/ml when rat serum was presented in the medium. Cefotaxime reduced titers of bacteria within abscesses after 5 days of therapy (5.77 +/- 0.68 log10 CFU/g) in comparison with those in control animals (7.38 +/- 0.28 log10 CFU/g, p less than 0.05). Moxalactam, the in vitro activity of which was not augmented by serum, proved ineffective in the animal model. While these observations do not have direct therapeutic relevance, they offer a possible explanation for the relatively infrequent occurrence of enterococcal superinfection in patients treated with cefotaxime.

Abscess↗
PubMed1992

In vivo activity of the combination of daptomycin and fosfomycin compared with daptomycin alone against a strain of Enterococcus faecalis with high-level gentamicin resistance in the rat endocarditis model.

The in vivo activity of the combination of daptomycin and fosfomycin against a beta-lactamase-producing, highly gentamicin-resistant strain of Enterococcus faecalis in a relapse model of rat endocarditis was studied. Minimum inhibitory concentrations (MICs) (micrograms per milliliter) for these agents against this strain were 4 (daptomycin) and 16 (fosfomycin). Time-kill studies demonstrated synergistic bactericidal activity when daptomycin (0.5 micrograms/ml) and fosfomycin (32 micrograms/ml) were combined. There was no significant difference between the number of valves sterilized by daptomycin alone [six (35%) of 17 valves sterilized] and daptomycin+fosfomycin [ten (59%) of 17 valves sterilized] p = 0.3. These results suggest that the in vitro bactericidal synergism demonstrable between these two agents against strains of enterococci will not necessarily translate into greater therapeutic efficacy in clinical infections.

Animals↗
PubMed1994

An old antibiotic for a new multiple-resistant Enterococcus faecium?

Enterococci have become an important cause of nosocomial infections and may demonstrate high-level resistance to multiple antibiotics. We present the case of a 68-year-old man with a history of small cell lung cancer, who developed bacteremia due to a strain of Enterococcus faecium. The isolate was resistant to multiple antibiotics including vancomycin, ampicillin, aminoglycosides, quinolones, and macrolides. The patient was successfully treated with doxycycline and removal of an infected central venous catheter.

Aged↗
PubMed1994

Molecular epidemiology of gastric colonization by Enterococcus faecalis in a surgical intensive care unit.

We applied restriction endonuclease analysis of genomic DNA using pulsed field gel electrophoresis (PFGE) to study gastric colonization with Enterococcus faecalis among patients hospitalized in the surgical intensive care unit (SICU). Isolates were obtained by culturing prospectively the gastric contents of 140 patients in the SICU. In addition, cultures of respiratory specimens were obtained daily and cultures of blood, normally sterile body fluids, wounds, and urine were obtained when indicated clinically. A total of 177 isolates were obtained from 45 patients. Concentrations of E. faecalis in gastric fluid ranged from 1 x 10(2) colony forming units (CFU)/ml to greater than 5 x 10(7) CFU/ml (mean 8.0 x 10(6) CFU/ml). Overall, 33 different DNA types were identified by PEGE. In examining strain variation among isolates obtained from multiple anatomic sites over time, we found that the same DNA type was recovered from gastric aspirates, sputum, and wounds in a given patient and that these strains were carried over time. In general, given individuals were colonized with their own unique DNA type; however, one DNA type (type C) was shared by 11 different patients, and seven DNA types were shared by two individuals each. These results demonstrate the potential importance of gastric colonization as a reservoir for nosocomial strains of E. faecalis in an SICU setting.

Colony Count, Microbial↗
PubMed1994

Enteric eradication of vancomycin-resistant Enterococcus faecium with oral bacitracin.

The emergence of vancomycin-resistant Enterococcus faecium (VREF) has produced a therapeutic dilemma. The colonization of the intestinal tract with VREF may predispose patients to infections by this organism and may contribute to its nosocomial spread. It is reasonable to attempt to eradicate VREF from colonized patients. The optimal regimen, however, is unknown and this study was designed to evaluate the efficacy of oral regimens of vancomycin and bacitracin for the elimination of VREF from the enteric tract. Enterococcal isolates were tested for susceptibilities to vancomycin, bacitracin, and ampicillin with median minimum inhibitory concentrations of > 512 micrograms/ml, 10 units/ml, and 128 micrograms/ml, respectively. All patients were given an initial trial of oral vancomycin 125 mg every 6 h for 10 days. Those who failed oral vancomycin were then given oral bacitracin 25,000 units every 6 h for 10 days due to its favorable in vitro activity. VREF was eradicated from the stools of 42% of patients (eight of 19) receiving oral vancomycin as compared with all eight patients receiving oral bacitracin (P < 0.01). The organism recurred in two bacitracin patients (25%) 8 and 20 days after completion of therapy. Whether prior vancomycin therapy predisposed patients to colonization by VREF was also examined. Ten (53%) of 19 patients had received prior vancomycin therapy before isolation of VREF from the stool. Our data suggest that oral bacitracin may be an effective alternative to commercially available oral vancomycin for the eradication of VREF from the enteric tract.

Administration, Oral↗
PubMed1996

Selection of Enterococcus faecium strains with stable and unstable resistance to the streptogramin RP 59500 using stepwise in vitro exposure.

Therapy of vancomycin-resistant enterococcal infections is an increasing problem for many large medical centers. One promising agent for use against Enterococcus faecium is RP 59500 (Quinupristin/Dalfopristin). To assess the potential for emergence of resistant strains during clinical trials with this new compound, we collected and tested 11 vancomycin-resistant and three vancomycin-susceptible E. faecium strains. The strains were exposed to doubling dilutions of RP 59500, beginning at drug concentrations ranging from 0.25 to 16 micrograms/ml agar. A saturated swab with approximately 3 x 10(7) organisms/ml was spread as a lawn on agar containing RP 59500 and incubated at 35 degrees C for 48 h. Growth on the highest drug-containing plate was used to prepare the inoculum for the next series of resistance-selection experiments. After the final passage, the range of the highest RP 59500 plate concentration with growth was 32-512 micrograms/ml (initial resistance frequency ranging from 1 x 10(-6) to > 1 x 10(-4). After the selection of resistant strains, the organisms were passed twice weekly on antimicrobial agent-free media to determine resistance stability in the absence of drug. Resistance in strains with an RP 59500 MIC of > or = 16 micrograms/ml was stable after repeated passage for 4 weeks. These results indicate that stable resistance to RP 59500 can be selected in E. faecium when the organism is exposed to increasing drug concentrations only slightly above the minimum inhibitory concentration (MIC). This stable resistance is seen in strains exhibiting an MIC > or = 16 micrograms/ml, and unstable in those with resistance where the RP 59500 MIC is less than eight times the original drug MIC. Our observation suggests more than one mechanism of resistance is likely present when stable resistance to RP 59500 develops.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Venous and capillary diffusion of Enterococcus faecalis during experimental bacteremia in the chicken].

Capillary and venous diffusion of bacteria were studied in chickens after intravenous inoculation with a strain of Enterococcus faecalis isolated from a case of septicaemia in a human neonate. One group of 13 chickens was inoculated with 10(8) bacteria/animal and another group of 20 chickens with 10(7) bacteria/animal. The level of bacteraemia in the first group of chickens was similar in capillary and venous blood. With 10(8) E. faecalis, the clearance of bacteria was very weak. The level of bacteraemia in the second group was significantly higher (after 180 min) in capillary than in venous blood, which suggests slower bacterial clearance in capillary than in venous blood. However, clearance was more efficient with the inoculum of 10(7) E. faecalis than with the first type of inoculum. These experimental data confirm the greater sensitivity of capillary rather than venous sampling, which we have already observed in chickens, under similar experimental conditions, with Escherichia coli K12. Persistence of bacteria in the capillaries could account for the higher level of sensitivity of capillary microblood culture than venous blood culture, which is in agreement with the capillary trapping hypothesis of Knudson and Alden.

Animals↗