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Extracellular proteinase from Enterococcus faecalis subsp. liquefaciens. I. Growth and extracellular proteinase production under different culture conditions.

Growth and extracellular proteinase production by Enterococcus faecalis subsp. liquefaciens was studied on several culture media and under different incubation conditions. The organisms grew well and developed extracellular proteinase activity on proteinaceous media, but when it grew on Collins basal medium (lacking of protein), growth was poor and proteinase activity was not detected. The activation energy for growth was estimated to be 116 kJ/mol, the optimum being at 37 degrees C. Proteinase production was not affected by temperature in the range studied (7-45 degrees C). Growth rate was not affected by aeration although a higher amount of microorganisms was observed on shaking the culture during incubation. Likewise, extracellular proteolytic activity was about twice higher in cultures shaken at 2.3 or 3.3 Hz than in those shaken at 0 or 1.3 Hz.

Bacteriological Techniques

Serum dependent expression of Enterococcus faecalis adhesins involved in the colonization of heart cells.

Our previous studies have shown that the adhesive ability of Enterococcus faecalis is dependent on the strain and is further modified by growth in serum. The data reported here demonstrate that E. faecalis adherence is mediated by carbohydrate residues present on the bacterial cell surface. Some of these (D-mannose and D-glucose) are expressed by strains isolated from both urinary tract infections (UTI) and endocarditis (EN) when the cells are grown in brain-heart infusion broth (BHIB), and mediate adherence to either urinary tract epithelial cells or the Girardi Heart (GH) cell line. Other residues are present only on EN strains (D-galactose and L-fucose) and mainly mediate adherence to GH cells. These ligands can also be expressed by UTI isolates after growth in serum. D-galactose-bearing adhesins also seem to be involved in internalization of serum grown UTI strains and BHIB or serum grown EN isolates into GH cells.

Bacterial Adhesion

Control of Enterococcus faecalis sex pheromone cAD1 elaboration: effects of culture aeration and pAD1 plasmid-encoded determinants.

Aeration of plasmid-free Enterococcus faecalis strains resulted in an 8- to 16-fold decrease in sex pheromone cAD1 activity in culture filtrates. Levels of two unrelated pheromones, cPD1 and cAM373, were unaffected by culture aeration. Aeration also resulted in a decrease in the expression of conjugative transfer functions observed in cells containing pAD1 traB mutations, verifying a link between traB function and pheromone "shutdown." Tests with a series of pAD1 mini-plasmids indicated that the product of the traB gene was involved in, but not sufficient for, pheromone shutdown; the cooperation of one or more other gene products encoded within the pheromone response control region was required.

Aerobiosis

[Activity of Enterococcus faecalis (FK-23) preparation as a biological response modifier].

The biological activity of a preparation of heat killed cells of Enterococcus faecalis, FK-23 which was isolated from the feces of a healthy human, was investigated in C3H/He mice. Intraperitoneal injection of the preparation caused an accumulation of neutrophils and macrophages in the peritoneal cavity of the mice 6 h later. As a parameter of the activation of macrophages, the effect of the FK-23 preparation on the production of tumor necrosis factor (TNF) was examined. The mice were given two consecutive intravenous injections of the preparation at a dose of 10 micrograms/mouse and, 3 h later, of 300 micrograms/mouse. The TNF level in the sera reached 99 U/ml in mice 2 h after the second injection. This preparation also stimulated peritoneal macrophages to produce TNF in vitro and increased the capacity of neutrophils to adhere to plastic plates and to release active oxygens, but did not induce blastogenic transformation of lymphocytes. These results suggest that the FK-23 preparation is a biological response modifier (BRM) with various activities on phagocytes similar to a streptococcal antitumor agent, OK432.

Animals

Comparison of the gentamicin resistance transposon Tn5281 with regions encoding gentamicin resistance in Enterococcus faecalis isolates from diverse geographic locations.

The genetic determinant encoding gentamicin resistance (Gmr) on the beta-lactamase encoding plasmid pBEM10 of Enterococcus faecalis HH22 is carried on a transposon, termed Tn5281, that is highly related to the staphylococcal Gmr transposons Tn4001 found in Australian isolates of Staphylococcus aureus and Tn4031 found in United States isolates of Staphylococcus epidermidis. We have now studied plasmid DNA from Gmr strains of E. faecalis isolated from diverse geographical locations (Houston, Pennsylvania, Thailand, and Chile) by using restriction endonuclease analysis and DNA-DNA hybridization to determine whether other Gmr E. faecalis carry Tn5281 or a similar type of element. We also compared these enterococci to several United States isolates of Staphylococcus aureus with nonmobile Gmr determinants. Three E. faecalis isolates (from Houston and Chile) carried Tn5281-like elements, whereas two isolates (from Houston and Pennsylvania) had restriction endonuclease and DNA-DNA hybridization patterns more similar to those of the Tn4001-IS257 hybrid found in the nonmobile Gmr determinants in United States isolates of S. aureus. A strain from Thailand had a third pattern unrelated to either Tn5281 or the nonmobile Gmr determinants present in United States isolates of S. aureus. Our results demonstrate that there is both similarity and diversity between the Gmr determinant of strains of E. faecalis isolated in diverse geographic locations.

Anti-Bacterial Agents

Relationship of the chemical structure and immunobiological activities of lipoteichoic acid from Streptococcus faecalis (Enterococcus hirae) ATCC 9790.

Two molecular species of lipoteichoic acid (LTA 1 and LTA 2) were isolated from whole cells of Streptococcus faecalis (Enterococcus hirae) ATCC 9790 by hydrophobic chromatography on Octyl Sepharose CL-4B. Chemical analysis revealed that LTA 1 and LTA 2 contained two and four acyl lipid anchors respectively. LTA 1 was less active than LTA 2 in inducing cytokines, except interleukin-1 (IL-1), but their in vivo antitumour effects were similar. LTA 2 was a potent inducer of tumour necrosis factor (TNF) and interferon (IFN) production and had excellent antitumour activity against Meth A fibrosarcoma established in mice. Deacylation of LTA 2 by alkaline hydrolysis abolished these biological activities. The phosphatidylglycolipid fraction derived from LTA 2 after acid hydrolysis could also induce TNF, IFN, and IL-1 production, as well as having antitumour activity against Meth A fibrosarcoma. Therefore, the lipid anchor portion of S. faecalis LTA may play an important role in the manifestation of these various biological activities.

Animals

Enterococcus faecalis: specific and non-specific interactions with human polymorphonuclear leukocytes.

In previous studies we have demonstrated that the ability of Enterococcus faecalis to adhere to and to be internalized in human urinary tract epithelial cells, Girardi Heart cells and human polymorphonuclear leukocytes (PMNs), was dependent on whether the strain had been isolated from urinary tract infections (UTI) or endocarditis (EN) respectively. These properties were further modified by growth of the organism in human serum. In the present report, using competition assays we show that adhesins containing a D-glucose moiety play a role in mediating the interactions between human PMNs and E. faecalis strains isolated from UTI and grown in brain-heart infusion broth (BHIB). On the other hand, adhesins containing both D-glucose and D-galactose moieties were involved in the interactions between PMNs and serum grown UTI isolates or EN isolates grown in either BHIB or human serum. Moreover, the impairment in the association between both UTI and EN strains after growth in serum appears to be at least partially related to a decrease in enterococcal surface hydrophobicity.

Adult

Genome-wide analysis of Enterococcus faecalis genes that facilitate interspecies competition with Lactobacillus crispatus.

Enterococci are opportunistic pathogens notorious for causing a variety of infections. While both Enterococcus faecalis and Lactobacillus crispatus are commensal residents of the vaginal tract, the molecular mechanisms that enable E. faecalis to take advantage of a vaginal biome with lower counts of lactobacilli to colonize the vaginal tract and induce aerobic vaginitis remain unknown. Here, we show that L. crispatus eradicates E. faecalis in a contact-independent manner. Using transposon sequencing to identify E. faecalis OG1RF transposon (Tn) mutants that are either under-represented or over-represented when co-cultured with L. crispatus, we found that Tn mutants with disruption in the dltABCD operon, that encodes the proteins responsible for the D-alanylation of teichoic acids, and OG1RF_11697 encoding for an uncharacterized hypothetical protein are more susceptible to killing by L. crispatus. Inversely, Tn mutants with disruption in ldh1, which encodes for L-lactate dehydrogenase, are more resistant to L. crispatus killing. Using the Galleria mellonella infection model, we show that co-injection of L. crispatus with E. faecalis OG1RF enhances larvae survival while this L. crispatus-mediated protection was lost in larvae co-infected with either L. crispatus and E. faecalisΔldh1 or Δldh1Δldh2 strains. Last, using RNA sequencing to identify E. faecalis genes that are differently expressed in the presence of L. crispatus, we found major changes in the expression of genes associated with glycerophospholipid metabolism, central metabolism, and general stress responses. The findings in this study provide insights into how E. faecalis mitigate assaults by L. crispatus.IMPORTANCEEnterococcus faecalis is an opportunistic pathogen notorious for causing a multitude of infections. As vaginal commensals, E. faecalis must interact with Lactobacillus crispatus, but how E. faecalis overcomes or mitigate assaults by L. crispatus killing remains unknown. We show that L. crispatus eradicates E. faecalis temporally in a contact-independent manner. Using high-throughput molecular approaches, we identified genetic determinants that enable E. faecalis to compete with L. crispatus. This study represents an important first step for the identification of adaptive genetic traits required for enterococci to tolerate assaults by lactobacilli.

Enterococcus faecalis

Factors influencing determination of high-level aminoglycoside resistance in Enterococcus faecalis.

The ability of seven methods to detect high-level gentamicin (58 strains) and streptomycin resistance (56 strains) among 107 Enterococcus faecalis isolates was investigated at the University of Chicago Medical Center and the University of Nebraska Medical Center. Methods included a standard agar screen plate, high-content disk diffusion, Remel (Lenexa, Kans.) EF Synergy Quad plates, standard microdilution panels prepared in house, Pasco MIC Gram-Positive panels (Difco Laboratories, Detroit, Mich.), MicroScan MIC Type 5 dry panels (Baxter Healthcare Corp., MicroScan Div., West Sacramento, Calif.), and Vitek GPS-TA cards (Vitek Systems Inc., Hazelwood, Mo.). Results indicating false resistance were not obtained by any method, and there was 100% agreement between the results of the disk diffusion and standard agar screen methods. Prolonging incubation from 24 to 48 h increased resistance detection for both agar and microdilution screens. EF Synergy Quad plates inoculated with micropipettes detected 100% of the streptomycin- and gentamicin-resistant isolates. Resistance detection for streptomycin and gentamicin, respectively, was 93 and 96% by standard microdilution, 93 and 98% by Pasco panels, 88 and 89% by MicroScan panels, and 88 and 91% by Vitek GPS-TA cards. False susceptibility occurred more frequently with streptomycin-resistant isolates than it did with gentamicin-resistant strains and appeared to be strain related in some instances. The use of an increased inoculum size enhanced resistance detection with these strains, but it complicated interpretation of results and led to the selection of streptomycin-resistant mutants. Until results of further studies delineate optimum test conditions, a delay in the final interpretation of agar and microdilution screen results until 48 h for isolates showing no or light growth at 24 h may help to minimize the occurrence of false susceptibility reporting.

Anti-Bacterial Agents

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

Evidence of incorporation of the chromosomal beta-lactamase gene of Enterococcus faecalis CH19 into a transposon derived from staphylococci.

We recently reported the chromosomal location of the staphylococcal beta-lactamase gene in four strains of Enterococcus faecalis. Transfer of this gene from strain CH19 to an enterococcal recipient was accompanied by transfer of numerous other antimicrobial resistance determinants in the absence of detectable plasmid DNA. A restriction map developed by comparing digestions of the regions surrounding the beta-lactamase gene in donor and recipient chromosomes resembles published maps of previously described staphylococcal beta-lactamase transposons, particularly in the area of the structural gene and its downstream region. In addition, DNA sequence analysis of the region immediately downstream of the beta-lactamase gene from both CH19 and its transcipient, CX19, revealed the presence of a 121-bp inverted repeat region found in Tn552 and Tn4002, two previously described staphylococcal beta-lactamase transposons. These results suggest that the chromosomal beta-lactamase gene of E. faecalis CH19 is incorporated into a transposonlike element derived from staphylococci.

Base Sequence

Isolation and characterisation of the pyruvate dehydrogenase complex of anaerobically grown Enterococcus faecalis NCTC 775.

In this contribution the isolation and some of the structural and kinetic properties of the pyruvate dehydrogenase complex (PDC) of anaerobically grown Enterococcus faecalis are described. The complex closely resembles the PDC of other Gram-positive bacteria and eukaryotes. It consists of four polypeptide chains with apparent molecular masses on SDS/PAGE of 97, 55, 42 and 36 kDa, and these polypeptides could be assigned to dihydrolipoyl transacetylase (E2), lipoamide dehydrogenase (E3) and the two subunits of pyruvate dehydrogenase (E1 alpha and E1 beta), respectively. The E2 core has an icosahedral symmetry. The apparent molecular mass on SDS/PAGE of 97 kDa of the E2 chain is extremely high in comparison with other Gram-positive organisms (and eukaryotes) and probably due to several lipoyl domains associated with the E2 chain. NADH inhibition is mediated via E3. The mechanism of inhibition is discussed in view of the high PDC activities in vivo that are found in E. faecalis, grown under anaerobic conditions.

Acetyltransferases

Conjugal transfer of Tn916, Tn916 delta E, and pAM beta 1 from Enterococcus faecalis to Butyrivibrio fibrisolvens strains.

Anaerobic filter matings of Butyrivibrio fibrisolvens H17c, CF3, D1, or GS113, representing different DNA relatedness groups, were done with Enterococcus faecalis CG110, which contains chromosomally inserted Tn916. Tetracycline-resistant transconjugants were obtained with each mating pair at average frequencies of 4.4 x 10(-6) (per recipient) and 5.2 x 10(-6) (per donor). The transfer frequencies of Tn916 into B. fibrisolvens varied 5- to 10-fold with mating time, strain, and growth stage. By using Southern hybridization with pAM120 as the probe, Tn916 was shown to insert at one or more separate chromosomal sites for each strain of B. fibrisolvens. Retransfer of Tn916 from B. fibrisolvens H17c or CF3 to E. faecalis OG1-X or JH 2-2 or to B. fibrisolvens D1 or GS113 could not be shown. Matings of E. faecalis RH110, which contains chromosomally inserted Tn916 delta E, with B. fibrisolvens 49, H17c, D1, CF3, GS113, or VV-1 resulted in erythromycin-resistant transconjugants at average frequencies of 5.3 x 10(-7) (per recipient) and 2.5 x 10(-7) (per donor). Tn916 delta E was shown by Southern hybridization with pAM120 to insert at one or more sites in the chromosome of each strain. B. fibrisolvens H17c was anaerobically filter mated with E. faecalis JH 2-SS, which contains pAM beta 1. Erythromycin-resistant transconjugants were obtained at frequencies of 2 x 10(-5) (per recipient) and 6 x 10(-5) (per donor). The presence of pAM beta 1 in these transconjugants could not be shown by agarose gel electrophoresis of plasmid minilysates but could be shown by Southern hybridization analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteroidaceae

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

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

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

Induction of autolysis in Enterococcus faecalis S-47 by peptide AS-48.

In addition to its bactericidal mode of action, the peptide antibiotic AS-48 exhibits a bacteriolytic effect on Enterococcus faecalis S-47 that is associated with autolysin activation. Bacteriolysis induced by the antibiotic can be modulated by addition of EDTA, divalent cations and autolysin activators (trypsin) or inhibitors (cardiolipin), suggesting that topologic regulation of the autolysins is involved in the process. In addition, inhibitors of protein and RNA synthesis interfere markedly with bacteriolysis, as do ionophores and the ATPase inhibitor DCCD, suggesting the participation of an internal messenger in autolysin activation in the presence of AS-48.

Anti-Bacterial Agents