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Susceptibility of Enterococcus faecalis to twelve antibiotics, time-kill assays, and high-level aminoglycoside resistance in a university hospital in Argentina.

A total of 201 Enterococcus faecalis strains isolated from different body sites were tested to (i) establish their antibiotic susceptibility pattern; (ii) determine the percentage of strains highly resistant (MIC greater than 2,000 micrograms/ml) to five aminoglycosides and (iii) know if the combination of penicillin or ampicillin plus an aminoglycoside is reliably synergistic for the strains with low-level resistance (MICs ranged from the break point of susceptibility for each aminoglycoside to 2,000 micrograms/ml). Erythromycin exhibited very poor activity with MIC90 greater than 128 micrograms/ml. Pefloxacin and norfloxacin had intermediate activity, inhibiting 50% of isolates at 4 micrograms/ml and 90% at 8 micrograms/ml. Trimethoprim-sulfamethoxazole (1:20) inhibited 94% of isolates at less than or equal to 2 micrograms/ml and 87.6% at less than or equal to 0.5 microgram/ml. Ampicillin, penicillin and piperacillin were the most potent agents studied. Ninety percent of strains were inhibited at 1 microgram/ml of ampicillin and 4 micrograms/ml of penicillin and piperacillin. The E. faecalis isolates were relatively or totally resistant to the aminoglycosides. Ninety six (47.8%) were highly resistant at least to one of them. High level resistance to streptomycin was found in 47.3% of all strains and was the most frequent resistance encountered; amikacin highly resistant strains were the less common and accounted for 4.5%. Low-level resistance to the aminoglycosides ranged from 50.2% (for streptomycin) to 94.5% (for amikacin). Thirty one E. faecalis isolates were selected for 24-time kill-assays. There was a good correlation between resistance to penicillin or ampicillin aminoglycoside synergy in all but 3 strains which were highly resistant. Among the strains with low-level resistance to the aminoglycosides, there was no synergy in 37 (63.8%) of 58 killing assays with each of the aminoglycosides combined with penicillin. These results demonstrate that the susceptibility to 2,000 micrograms/ml of the aminoglycoside does not assure the penicillin or ampicillin aminoglycoside synergism.

Aminoglycosides↗

Nucleotide sequence of the beta-lactamase gene from Enterococcus faecalis HH22 and its similarity to staphylococcal beta-lactamase genes.

The nucleotide sequence of the constitutively produced beta-lactamase (Bla) gene from Enterococcus faecalis HH22 was shown to be identical to the published sequences of three of four staphylococcal type A beta-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred forty nucleotides upstream of the beta-lactamase start codon were determined for an inducible staphylococcal beta-lactamase and were identical to those of the constitutively expressed enterococcal gene, indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The genes for the enterococcal Bla and an inducible staphylococcal Bla were each cloned into a shuttle vector and transformed into enterococcal and staphylococcal recipients. The major difference between the backgrounds of the two hosts was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene. The location of the enzyme was found to be host dependent, since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell-bound enzyme in the enterococcus. On the basis of the identities of the enterococcal Bla and several staphylococcal Bla sequences, these data suggest the recent spread of beta-lactamase to enterococci and also suggest the loss of a functional repressor.

Base Sequence↗

Turbidimetric response of Staphylococcus aureus and Enterococcus faecalis to daptomycin.

The response to daptomycin of three strains of Staphylococcus aureus (one of which was methicillin-resistant) and a strain of Enterococcus faecalis was investigated by continuous turbidimetric monitoring. Daptomycin caused partial inhibition of bacterial growth at concentrations well below those suppressing growth overnight. The activity of the drug was more than 200-fold greater in the presence of a physiological concentration of calcium (2.5 mmol/l) than in its absence. Variants present in cultures inhibited by daptomycin were shown to exhibit decreased susceptibility to the drug and the susceptibility declined further on repeat exposure; however, there was a slow but incomplete reversion to susceptibility on sequential subculture in drug-free broth. Resistance was difficult to induce when the concentration of calcium in the broth exceeded 2.5 mmol/l.

Anti-Bacterial Agents↗

Isolation of Enterococcus faecalis clinical isolates that efficiently adhere to human bladder carcinoma T24 cells and inhibition of adhesion by fibronectin and trypsin treatment.

The adherence of Enterococcus faecalis strains to human T24 cells was examined by scanning electron microscopy. Five highly adhesive strains were identified from 30 strains isolated from the urine of patients with urinary tract infections. No efficiently adhesive strains were found among the 30 strains isolated from the feces of healthy students. The five isolated strains also adhered efficiently to human bladder epithelial cells. Analysis of restriction endonuclease-digested plasmid DNAs and chromosome DNAs showed that the five strains were different strains isolated from different patients. The adhesiveness of these strains was inhibited by treatment with fibronectin or trypsin, implying that a specific protein (adhesin) on the bacterial cell surface mediates adherence to fibronectin on the host cell surfaces, and the adhesin differs from the reported adhesins.

Bacterial Adhesion↗

The Enterococcus faecalis sigV protein is an extracytoplasmic function sigma factor contributing to survival following heat, acid, and ethanol treatments.

Analysis of the genome sequence of Enterococcus faecalis allowed the identification of two genes whose protein products showed 33 and 34% identity with those of sigV and yrhM of Bacillus subtilis, respectively. These genes, named sigV and rsiV, are predicted to encode members of the extracytoplasmic function subfamily of eubacterial RNA polymerase sigma and anti-sigma factors, respectively. This group of sigma factors has been shown to regulate gene expression in response to stress conditions. sigV and rsiV were shown to be under the control of the same promoter. The transcriptional start site was determined, and the 1.5-kb mRNA transcript was shown to be overexpressed under glucose and complete starvation, as well as under physicochemical treatments. Three mutants, affected in sigV, rsiV, and both genes, were constructed by double-crossover recombination within the genome of E. faecalis strain JH2-2. Compared with the wild type and the rsiV mutant, the sigV mutants were more susceptible to heat shock, acid, and ethanol treatments and displayed decreased survival during long-term starvation. A nisin-inducible sigV gene construction used in complementation assays restored the wild phenotype of the sigV mutants, confirming the involvement of SigV in the heat shock, ethanol, and acid stress responses. Northern blot analysis carried out with the three mutant strains revealed the inhibition of sigV expression by the related anti-sigma factor gene rsiV. In addition, putative candidates of the sigV regulon determined by computer search for the sigV promoter sequence were analyzed.

Amino Acid Sequence↗

Enterococcus faecalis: an unusual cause of meningitis in HIV-infected patients.

A case of a severely immunosuppressed HIV-infected man with meningitis caused by Enterococcus faecalis is presented. E faecalis is a distinctly unusual cause of bacterial meningitis in patients with HIV infection and should be considered in the differential diagnosis of purulent meningitis in such patients to ensure optimal empiric treatment.

AIDS-Related Opportunistic Infections↗

Conjugative transfer of Tn916 in Enterococcus faecalis: trans activation of homologous transposons.

Tn916 [carries tet(M)] is a 16.4-kb conjugative transposon that can establish itself in multiple copies in Enterococcus faecalis. To study the interaction of coresident homologous transposons during conjugation, an E. faecalis mutant defective in homologous recombination was utilized for construction of strains harboring Tn916 delta E (a derivative in which erm is substituted for tet) on the chromosome and Tn916 on a nonconjugative plasmid. When these strains were used as donors, the two transposons were able to transfer independently; however, they were found to transfer and become coestablished in the recipient up to 50% of the time. In contrast, cotransfer of a plasmid marker located outside the transposon occurred at a frequency of no greater than 0.5%. Separate experiments showed that mobilization of the nonconjugative plasmids pAM401 and pVA749 by chromosome-borne copies of Tn916 occurred only at low frequencies (generally less than 2% cotransfer). The data imply that the initiation of transposition of Tn916 results in a trans activation that is specific for homologous transposons present in the same cell.

Chromosomes, Bacterial↗

Changes in arterial blood pressure in hypertensive rats caused by long-term intake of milk fermented by Enterococcus faecalis CECT 5728.

We have evaluated the changes in arterial blood pressure caused in spontaneously hypertensive rats (SHR) by long-term intake of an Enterococcus faecalis CECT 5728-fermented milk with significant angiotensin-converting enzyme (ACE)-inhibitory activity. After being weaned, male 3-week-old SHR were randomized into five groups. Until the 20th week of life, rats in each group were given one of the following drinking fluids: tap water (negative control 1), a fermented milk without ACE-inhibitory activity (negative control 2), captopril (100 mg/kg) (positive control), the E. faecalis CECT 5728-fermented milk that had significant ACE-inhibitory activity, or Ca-enriched E. faecalis CECT 5728-fermented milk. Animals in the different groups were then given tap water as drinking fluid from the 20th to 25th week of life. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured weekly in the rats, from the 6th to 25th week of life, by the tail-cuff method. A definite decrease in SBP and DBP could be observed in the rats treated with captopril and also in the rats that received the E. faecalis CECT 5728-fermented milks. The greatest antihypertensive effect was observed when the pharmacological treatment was administered. The effect of the Ca-enriched fermented milk was slightly more accentuated and more constant than the effect of the E. faecalis CECT 5728-fermented milk that had not been enriched in Ca. SBP and DBP increased in the treated SHR when the corresponding antihypertensive treatment was removed. Fermentation of milk with E. faecalis CECT 5728 may therefore be a successful strategy to produce a functional food with antihypertensive activity.

Angiotensin-Converting Enzyme Inhibitors↗

Studies on the distribution of high-level gentamicin-resistant Enterococcus faecalis using ribotyping and restriction analysis of the 16S-23S rRNA intergenic spacer sequences.

Twenty-five high-level gentamicin resistant (HLGR) Enterococcus faecalis strains were isolated from three different University laboratories in Italy. The resistant strains were variously distributed in the three centers with percentages of prevalence ranging from about 3% up to 14%. Almost all strains shared high-level resistance to streptomycin (23 out of 25). Ribotyping and restriction analysis of the 16S-23S rRNA intergenic spacer sequences were used to genetically differentiate the various strains and to study their spreading in the university hospitals serviced by the three laboratories. At least three ribotypes were identified, which showed a peculiar distribution in the various centers. Only the ribotype B was isolated from the University of Padua. In Cagliari, most strains belonged to ribotype A (4/6), whereas in Genoa there was an equal distribution of the ribotypes A and B. A clonal spreading of some HLGR strains is suggested by these findings. The restriction analysis of the 16S-23S rRNA intergenic-spacer sequences gave comparable results with classical ribotyping and, in addition, was quicker and easier to perform than the latter.

Drug Resistance, Microbial↗

Efficacy of ampicillin plus ceftriaxone in treatment of experimental endocarditis due to Enterococcus faecalis strains highly resistant to aminoglycosides.

The purpose of this work was to evaluate the in vitro possibilities of ampicillin-ceftriaxone combinations for 10 Enterococcus faecalis strains with high-level resistance to aminoglycosides (HLRAg) and to assess the efficacy of ampicillin plus ceftriaxone, both administered with humanlike pharmacokinetics, for the treatment of experimental endocarditis due to HLRAg E. faecalis. A reduction of 1 to 4 dilutions in MICs of ampicillin was obtained when ampicillin was combined with a fixed subinhibitory ceftriaxone concentration of 4 micrograms/ml. This potentiating effect was also observed by the double disk method with all 10 strains. Time-kill studies performed with 1 and 2 micrograms of ampicillin alone per ml or in combination with 5, 10, 20, 40, and 60 micrograms of ceftriaxone per ml showed a > or = 2 log10 reduction in CFU per milliliter with respect to ampicillin alone and to the initial inoculum for all 10 E. faecalis strains studied. This effect was obtained for seven strains with the combination of 2 micrograms of ampicillin per ml plus 10 micrograms of ceftriaxone per ml and for six strains with 5 micrograms of ceftriaxone per ml. Animals with catheter-induced endocarditis were infected intravenously with 10(8) CFU of E. faecalis V48 or 10(5) CFU of E. faecalis V45 and were treated for 3 days with humanlike pharmacokinetics of 2 g of ampicillin every 4 h, alone or combined with 2 g of ceftriaxone every 12 h. The levels in serum and the pharmacokinetic parameters of the humanlike pharmacokinetics of ampicillin or ceftriaxone in rabbits were similar to those found in humans treated with 2 g of ampicillin or ceftriaxone intravenously. Results of the therapy for experimental endocarditis caused by E. faecalis V48 or V45 showed that the residual bacterial titers in aortic valve vegetations were significantly lower in the animals treated with the combinations of ampicillin plus ceftriaxone than in those treated with ampicillin alone (P < 0.001). The combination of ampicillin and ceftriaxone showed in vitro and in vivo synergism against HLRAg E. faecalis.

Algorithms↗

Contribution of VanY D,D-carboxypeptidase to glycopeptide resistance in Enterococcus faecalis by hydrolysis of peptidoglycan precursors.

The vanR, vanS, vanH, vanA, and vanX genes of enterococcal transposon Tn1546 were introduced into the chromosome of Enterococcus faecalis JH2-2. Complementation of this portion of the van gene cluster by a plasmid encoding VanY D,D-carboxypeptidase led to a fourfold increase in the vancomycin MIC (from 16 to 64 micrograms/ml). Multicopy plasmids pAT80 (vanR vanS vanH vanA vanX) and pAT382 (vanR vanS vanH vanA vanX vanY) conferred similar levels of vancomycin resistance to JH2-2. The addition of D-alanine (100 mM) to the culture medium restored the vancomycin susceptibility of E. faecalis JH2-2/pAT80. The pentapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala partially replaced pentadepsipeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac when the strain was grown in the presence of D-alanine. In contrast, resistance mediated by pAT382 was almost unaffected by the addition of the amino acid. Expression of the vanY gene of pAT382 resulted in the formation of the tetrapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala, indicating that a portion of the cytoplasmic precursors had been hydrolyzed. These results show that VanY contributes to glycopeptide resistance in conditions in which pentapeptide is present in the cytoplasm above a threshold concentration. However, the contribution of the enzyme to high-level resistance mediated by Tn1546 appears to be moderate, probably because hydrolysis of D-alanyl-D-alanine by VanX efficiently prevents synthesis of the pentapeptide.

Alanine↗

Bactericidal activity of antibiotics alone and in combination against Enterococcus faecalis in a pharmacodynamic model.

We evaluated the bactericidal kinetics of teicoplanin, mezlocillin, netilmicin, and ciprofloxacin alone and in dual combinations against strains of Enterococcus faecalis susceptible or resistant to ampicillin in a pharmacodynamic model reproducing in bacterial culture in active human plasma or Mueller-Hinton broth the serum kinetics of these antibiotics in humans. Killing was not different in cultures grown in plasma and those grown in broth. Antibiotics used alone had no or low bactericidal activity except for high-dose ciprofloxacin (600 mg intravenously [iv] twice daily or 750 mg orally twice daily), which achieved a 3- to 4-log reduction in colony-forming units (cfu). Netilmicin was equally active at 6 mg/kg once a day or 2 mg/kg three times daily in all combinations. No major increase in bactericidal activity was detected in any combination that included mezlocillin. Maximal synergistic killing was observed for the combination of teicoplanin plus netilmicin (both at three-times daily and once-daily dosing), which sterilized the bacterial cultures (initial inoculum, 10(6) cfu/mL). Combinations of ciprofloxacin at 600 mg iv twice daily and 750 mg orally twice daily plus either teicoplanin or netilmicin were less synergistic but equally effective in total killing as a result of the high bactericidal activity of ciprofloxacin alone.

Anti-Bacterial Agents↗

Repetitive sequence-based PCR versus pulsed-field gel electrophoresis for typing of Enterococcus faecalis at the subspecies level.

Repetitive sequence-based PCR was compared to pulsed-field gel electrophoresis (PFGE) for the ability to discriminate Enterococcus faecalis isolates at the subspecies level. The BOXA2R primer, derived from repetitive sequences in Streptococcus pneumoniae, was applied to 41 isolates of E. faecalis collected from various sources. The REP1R-Dt and REP2-Dt primers, derived from the gram-negative repetitive extragenic palindromic element, were also applied to 18 selected isolates. Of the 41 isolates examined, 7 were beta-lactamase producing and 8 were vancomycin resistant. By PFGE, 17 isolates had distinct patterns; the other 24 were classified into eight different clonal groups. By PCR using the BOXA2R primer, 16 isolates generated distinct patterns; the other 25 were classified into nine different clonal groups. There were only minor differences in the PCR results obtained by using the BOXA2R primer and the REP1R-Dt and REP2-Dt primers. Two isolates among vancomycin-resistant enterococci from the greater Houston, Tex., area were related by PFGE, distinct by PCR with the BOXA2R primer, and related by PCR with the REP1R-Dt and REP2-Dt primers. Clonal relationships among the remaining 39 isolates were similar by both PFGE and PCR. PCR reliably discriminated all epidemiologically unrelated isolates. Although PCR is less time consuming than PFGE, PCR results were more difficult to interpret than PFGE results, perhaps because fewer bands were generated by PCR than by PFGE and some PCR products were inconsistently seen.

Bacterial Typing Techniques↗

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↗

Insertions of IS256-like element flanking the chromosomal beta-lactamase gene of Enterococcus faecalis CX19.

We have previously identified an inverted repeat characteristic of staphylococcal beta-lactamase transposons adjacent to the chromosomal beta-lactamase genes of Enterococcus faecalis CH19 and its beta-lactamase-producing transconjugant CX19. Nucleotide sequence analysis of the CH19 beta-lactamase structural gene (blaZ) reveals it to be identical to the blaZ gene from E. faecalis HH22 and to the blaZ gene from the staphylococcal beta-lactamase transposon Tn552. We also report the presence of nucleotide sequence identical to a 317-bp region of the staphylococcal insertion sequence IS256 upstream of the blaZ gene in both CH19 and CX19. The identical segment of IS256 is present downstream of the blaZ gene of CX19, suggesting a second insertion of the element (in the inverted orientation) accompanying transfer to the recipient strain. Restriction analysis of the areas beyond the ClaI sites used to clone these regions suggests that full copies of the IS256-like element (designated IS256E) are present in all positions but that these elements were not directly involved in the transfer of the beta-lactamase gene to the recipient strain. We have also identified a region downstream of the second IS256E insertion site which exhibits substantial homology to ISSIW, an iso-ISSI insertion originally identified in Lactococcus lactis subsp. cremoris. These data suggest that the two enterococcal blaZ genes sequenced to date evolved from a common ancestor and may at one time have been incorporated into a transposon similar to Tn552. They also suggest that IS256-like elements are mobile in E. faecalis and capable of inserting in a manner consistent with the formation of novel composite transposons. Finally, they provide the first confirmation of the presence of an ISSI-like element in enterococci, raising the possibility that these elements play a role in the exchange of chromosomal antimicrobial resistance determinants.

Base Sequence↗

Infection-derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein.

We report the identification of a new cell wall-associated protein of Enterococcus faecalis. Studies on the distribution of the gene encoding this novel surface protein, Esp, reveal a significant (P < 0.001) enrichment in infection-derived E. faecalis isolates. Interestingly, the esp gene was not identified in any of 34 clinical E. faecium isolates or in 4 other less pathogenic enterococcal species tested. Analysis of the structural gene among various E. faecalis isolates reveals the existence of alternate forms of expression of the Esp protein. The deduced primary structure of the Esp protein from strain MMH594, inferred to be 1,873 amino acids (aa) with a predicted mass of approximately 202 kDa, reveals a core region consisting of repeat units that make up 50% of the protein. Esp bears global organizational similarity to the Rib and C alpha proteins of group B streptococci. Identity among Esp, Rib, and C alpha proteins is strikingly localized to a stretch of 13 aa within repeats of similar length. The high degree of conservation of this 13-residue sequence suggests that it plays an important role in the natural selection for this trait among infection-derived E. faecalis and group B streptococcal isolates.

Adolescent↗

Clinical and molecular epidemiology of Enterococcus faecalis bacteremia, with special reference to strains with high-level resistance to gentamicin.

The objective of this study was to characterize the clinical and molecular epidemiology of Enterococcus faecalis bacteremia, specifically that involving strains with high-level resistance to gentamicin (HLGR). Episodes of E. faecalis bacteremia at the Buffalo Veterans Affairs Medical Center from January 1986 to September 1989 were retrospectively identified. Of 94 episodes, 45 (48%) were due to strains with HLGR. Hemolytic activity was detected with greater frequency (85%) among the latter strains than among those without HLGR (P < .001). Of the 54 episodes for which medical charts were available for review, 94% were hospital acquired, and 46% were due to strains with HLGR. An examination of the plasmid DNA content of isolates revealed restriction-fragment-length polymorphism. One plasmid pattern was identified in 15 isolates with HLGR (P < .001), and chromosomal DNA digest patterns suggested a common clonality; there was no direct evidence for patient-to-patient spread of these strains. The use of antibiotics, the presence of invasive devices, surgery, and admission to an intensive-care unit were not significantly associated with HLGR bacteremia. Mortality during hospitalization was 65%, with no difference between figures for HLGR and non-HLGR infections. This high mortality regardless of gentamicin susceptibility status suggests that E. faecalis bacteremia is a marker for severe illness. In contrast to previous studies, this investigation identified no clinical factors associated with HLGR E. faecalis bacteremia.

Bacteremia↗

Role of the Enterococcus faecalis GelE protease in determination of cellular chain length, supernatant pheromone levels, and degradation of fibrin and misfolded surface proteins.

Gelatinase (GelE), a secreted Zn-metalloprotease of Enterococcus faecalis, has been implicated as a virulence factor by both epidemiological data and animal model studies. Expression of gelE is induced at a high cell density by the fsr quorum-sensing system. In the present study, GelE was shown to be responsible for the instability of a number of Asc10 (aggregation substance) mutant proteins, implying that GelE functions to clear the bacterial cell surface of misfolded proteins. Disruption of GelE production led to increased cell chain length of E. faecalis, from a typical diplococcus morphology to chains of 5 to 10 cells. This function of GelE was also exhibited when the protein was expressed in Streptococcus pyogenes. GelE-expressing E. faecalis strains were more autolytic, suggesting that GelE affects chain length through activation of an autolysin. GelE was also essential for degradation of polymerized fibrin. GelE expression reduced the titer of cCF10, the peptide pheromone that induces conjugation of pCF10, and pCF10 had increased conjugation into non-GelE-expressing strains. These new functions attributed to GelE suggest that it acts to increase the dissemination of E. faecalis in high-density environments.

Bacterial Proteins↗