Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence

Three agr-like genes (fsrA, fsrB, and fsrC, for Enterococcus faecalis regulator) were found upstream of the previously reported gelatinase gene (gelE) and a downstream putative serine protease gene (sprE; accession number Z12296) of Enterococcus faecalis OG1RF. The deduced amino acid sequence of fsrA shows 26% identity and 38% similarity to Staphylococcus aureus AgrA (the response regulator of the accessory gene regulator system in the agr locus), FsrB shows 23% identity and 41% similarity to S. aureus AgrB, and FsrC shows 23% identity and 36% similarity to S. aureus AgrC (the sensor transducer of Agr system). Northern blot analysis suggested that gelE and sprE are cotranscribed and that fsrB and fsrC are also cotranscribed in OG1RF. Northern blot analysis of fsrA, fsrB, fsrC, gelE, and sprE insertion mutants showed that fsrB, fsrC, gelE, and sprE are not expressed in fsrA, fsrB, and fsrC mutants, while insertion in an open reading frame further upstream of fsrA did not effect the expression of these genes, suggesting that agr-like genes may be autoregulated and that they regulate gelE and sprE expression, as further confirmed by complementation of fsr gene mutations with a 6-kb fragment which contains all three fsr genes in the shuttle vector, pAT18. Testing of 95 other isolates of E. faecalis showed that 62% produced gelatinase (Gel(+)), while 91% (including all Gel(+) strains) hybridized to a gelE probe; 71% (including all Gel(+) strains) hybridized to an fsr probe, corroborating the conclusion that both gelE and fsr are necessary for gelatinase production. Testing of fsrA, fsrB, and sprE mutants in a mouse peritonitis model showed that sprE and agr-like gene mutants resulted in highly significantly prolonged survival compared to the parent strain OG1RF, a finding similar to what we had previously shown for a gelE mutant. These results suggest that sprE and agr-like genes contribute to the virulence of E. faecalis OG1RF in this model.     

Prevalence of the fsr locus in Enterococcus faecalis infections

The fsr locus of Enterococcus faecalis confers virulence in animal models. A retrospective analysis of fsr prevalence in diverse E. faecalis clinical isolates demonstrated fsr in all endocarditis isolates versus 53% of stool isolates (P = 0.005). This supports a role for fsr-mediated virulence in the pathogenesis of enterococcal infections in humans.     

Contribution of gelatinase, serine protease, and fsr to the pathogenesis of Enterococcus faecalis endophthalmitis

Gelatinase and serine protease were found to contribute in concert to pathogenesis in a rabbit model of endophthalmitis. However, a mutant defective in the fsr regulator was observed to be more attenuated than a mutant rendered defective in the expression of gelatinase and serine protease as the result of a polar transposon insertion into the former. This increased attenuation suggests that there are possible additional pleiotropic effects of the defect in fsr on expression of traits contributing to the pathogenesis of enterococcal infection.     

Virulence effect of Enterococcus faecalis protease genes and the quorum-sensing locus fsr in Caenorhabditis elegans and mice

The expression of two Enterococcus faecalis extracellular virulence-related proteins, gelatinase (GelE) and serine protease (SprE), has been shown to be positively regulated by the fsr quorum-sensing system. We recently developed a novel system for studying E. faecalis pathogenicity that involves killing of the nematode worm Caenorhabditis elegans and showed that an E. faecalis fsrB mutant (strain TX5266) exhibited attenuated killing. We explore here the role of the fsr/gelE-sprE locus in pathogenicity by comparing results obtained in the nematode system with a mouse peritonitis model of E. faecalis infection. Insertion mutants of fsrA (TX5240) and fsrC (TX5242), like fsrB (TX5266), were attenuated in their ability to kill C. elegans. A deletion mutant of gelE (TX5264) and an insertion mutant of sprE (TX5243) were also attenuated in C. elegans killing, although to a lesser extent than the fsr mutants. Complementation of fsrB (TX5266) with a 6-kb fragment containing the entire fsr locus restored virulence in both the nematode and the mouse peritonitis models. The fsr mutants were not impaired in their ability to colonize the nematode intestine. These data show that extracellular proteases and the quorum-sensing fsr system are important for E. faecalis virulence in two highly divergent hosts: nematodes and mice.     

Decreased virulence of a gls24 mutant of Enterococcus faecalis OG1RF in an experimental endocarditis model

In the current study, the gls24 disruption mutant TX10100, previously shown to be more sensitive to bile salts and attenuated in a mouse peritonitis model, showed an approximately fivefold higher 50% infective dose than wild-type OG1RF in a rat endocarditis model. When administered as a mixture, TX10100, unlike a downstream glsB mutant, was significantly outnumbered by OG1RF in vegetations, organs, and blood, despite being inoculated in greater numbers. These results indicate that gls24 is important in the pathogenesis of enterococcal endocarditis.     

Molecular epidemiology of the fsr locus and of gelatinase production among different subsets of Enterococcus faecalis isolates

We examined 215 Enterococcus faecalis isolates and found that neither the two-component regulatory locus fsr (E. faecalis regulator) nor gelatinase production was more common in disease-associated isolates than in isolates colonizing healthy individuals (ca. 60 to 65%). The majority of gelatinase-negative isolates, including 14 endocarditis isolates (of 80 isolates tested), contained the previously described 23.9-kb deletion and lacked fsrA and fsrB. While these findings indicate that neither fsr nor gelatinase is required for E. faecalis to cause infection, this study did not address whether fsr or gelatinase affects the severity of disease, as it does in animal models.     

The Enterococcus faecalis fsrB gene,a key component of the fsr quorum-sensing system,is associated with virulence in the rabbit endophthalmitis model

We used a rabbit endophthalmitis model to explore the role of fsrB, a gene required for the function of the fsr quorum-sensing system of Enterococcus faecalis, in pathogenicity. A nonpolar deletion mutant of fsrB had significantly reduced virulence compared to wild type. Complementation of mutation restored virulence. These data corroborate the role of fsrB in E. faecalis pathogenesis and suggest that the rabbit endophthalmitis model can be used to study the in vivo role of quorum sensing.     

Virulence factors in isolates of Enterococcus faecalis from infective endocarditis and from the normal flora

Infective endocarditis (IE) is a severe infection associated with significant morbidity and mortality. Enterococcus faecalis is among the most common causes of this disease, and yet little is known about the pathogenesis of E. faecalis IE. We screened 21 E. faecalis isolates from IE and 21 isolates from normal flora for the putative virulence factors ace, asa1, gelE, and esp with PCR. In addition, we determined the ability of the isolates to form biofilm and to aggregate platelets. With the exception of biofilm formation, which was more pronounced in the normal flora group, there was no difference between the groups, indicating that many isolates have virulence properties and that host factors might determine if E. faecalis causes IE.     

Outbreak of infection with high-level gentamicin-resistant Enterococcus faecalis (HLGRE) in a Norwegian hospital

Objectives  To examine and characterize a suspected outbreak of high-level gentamicin-resistant Enterococcus (HLGRE) infection.
Methods  Eighty-nine patients with clinical infection diagnosed during hospital stay or within 30 days after discharge in the period from June 1995 to 31 December 1999 were included in the study. One control patient was assigned for each HLGRE patient according to localization in the hospital (same ward), time of admission (±3 months), and age (±10 years). Unadjusted risk analysis and multivariate logistic regression analysis were performed. Sixty-nine HLGRE strains were subjected to PCR amplification of the genes coding for aminoglycoside-3'- O -phosphoryltransferase-III (APH(3')-III) and aminoglycoside-6'- N -acetyltransferase/2'- O -phosphoryltransferase-III (AAC(6')/APH(2')).
Results  The gene aacA/aphD , associated with HLGRE, was detected by PCR in all isolates, and the gene aphA3 , associated with high-level streptomycin, kanamycin and amikacin resistance, was detected in 56 of the 69 isolates. None of the 69 isolates was resistant to glycopeptides or ampicillin. Resistance to ciprofloxacin was found in 57 (82.6%). Pulsed-field gel electrophoresis analysis revealed 12 different genotypes, among which two major clusters dominated.
Conclusions  Both clonal expansion and the emergence of unique strains contributed to the increased number of infections caused by HLGRE. Urinary catheterization, duration of hospital stay and antibiotic therapy were significant risk factors for HLGRE infection.     

Role of granulocytes in the induction of an experimental endocarditis with a dextran-producing Streptococcus sanguis and its dextran-negative mutant

The role of granulocytes in the induction of endocarditis with a dextran-producing Streptococcus sanguis and a dextran-negative mutant of this strain was studied. The number of colony-forming units of Streptococcus sanguis needed to colonize the vegetations in 50% of the rabbits (ID50) was significantly lower for the parent strain than for the dextran-negative mutant. However, in granulocytopenic rabbits the ID50s of both strains did not differ measurably. Dextran-negative streptococci were more readily cleared from the circulation than dextran-positive, but in this respect no difference was found between control and granulocytopenic rabbits, which indicates that clearance cannot account for the difference in ID50 between the two strains in the control group. At serum concentrations of 5% and lower, in-vitro granulocytes phagocytosed the dextran-negative streptococci more rapidly than the dextran-positive. The intracellular killing of the streptococci was no influenced by dextran production. This study suggests that an impaired phagocytic removal of attached bacteria from the vegetational surface can be a factor promoting the induction of endocarditis by dextran-producing streptococci.     

Detection of the macrolide-efflux protein A gene mef(A) in Enterococcus faecalis

The mef(A) gene codes for an efflux protein that conveys resistance to 14- and 15-membered macrolides. Enterococci are emerging pathogens, as well as indicator and reservoir bacteria that are known to have a strong tendency to acquire resistance genes. A total of 485 Enterococcus faecalis strains of porcine (n = 239) and human origin (n = 246) were screened for the presence of the mef(A) gene by using polymerase chain reaction. In total, 29 E. faecalis of porcine (n = 10) and human (n = 19) origin were positive for the presence of the mef(A) gene. Most of the mef(A)-containing strains were isolated from fecal samples of healthy individuals; only one strain originated from a stool sample of a diseased pig. To our knowledge, this is the first report on the occurrence of the mef(A) gene in E. faecalis apart from mating experiments. The main clinical relevance of this study is that donor E. faecalis might transfer the mef(A) gene to recipients that are usually combated with macrolides. Hence, the role of E. faecalis as a resistance reservoir with respect to limited treatment options are a cause for concern.     

High incidence of hemolysin production by Enterococcus (Streptococcus) faecalis strains associated with human parenteral infections.

Hemolysin production, clumping (pheromone) response, transferability of the hemolytic trait, and drug resistance were examined in 97 clinical isolates of Enterococcus (Streptococcus) faecalis. The isolates were derived from various sources (i.e., urine, pus, vagina, sputum, bile, and blood), and approximately 60% were found to be hemolytic. About 85% of the hemolytic strains exhibited a clumping response, compared with about 49% of the nonhemolytic strains. Over 50% of the hemolytic strains carried transferable hemolysin determinants, and in no case were drug resistance genes linked. The hemolytic strains exhibited multiple drug resistance more frequently than did the nonhemolytic strains. In contrast to the high frequency of hemolysin producers among parenteral isolates, strains derived from fecal specimens of healthy individuals exhibited a low (17%) incidence of hemolysin production.     

Role of granulocytes in the induction of an experimental endocarditis with a dextran-producing Streptococcus sanguis and its dextran-negative mutant.

The role of granulocytes in the induction of endocarditis with a dextran-producing Streptococcus sanguis and a dextran-negative mutant of this strain was studied. The number of colony-forming units of Streptococcus sanguis needed to colonize the vegetations in 50% of the rabbits (ID50) was significantly lower for the parent strain than for the dextran-negative mutant. However, in granulocytopenic rabbits the ID50s of both strains did not differ measurably. Dextran-negative streptococci were more readily cleared from the circulation than dextran-positive, but in this respect no difference was found between control and granulocytopenic rabbits, which indicates that clearance cannot account for the difference in ID50 between the two strains in the control group. At serum concentrations of 5% and lower, in-vitro granulocytes phagocytosed the dextran-negative streptococci more rapidly than the dextran-positive. The intracellular killing of the streptococci was no influenced by dextran production. This study suggests that an impaired phagocytic removal of attached bacteria from the vegetational surface can be a factor promoting the induction of endocarditis by dextran-producing streptococci.     

Antibodies to a surface-exposed, N-terminal domain of aggregation substance are not protective in the rabbit model of Enterococcus faecalis infective endocarditis

The aggregation substance (AS) surface protein from Enterococcus faecalis has been implicated as an important virulence factor for the development of infective endocarditis. To evaluate the role of antibodies specific for Asc10 (the AS protein from the conjugative plasmid pCF10) in protective immunity to infective endocarditis, an N-terminal region of Asc10 lacking the signal peptide and predicted to be surface exposed (amino acids 44 to 331; AS(44-331)) was cloned with a C-terminal histidine tag translational fusion and expressed from Escherichia coli. N-terminal amino acid sequencing of the purified protein revealed the correct sequence, and rabbit polyclonal antisera raised against AS(44-331) reacted specifically to Asc10 expressed from E. faecalis OG1SSp, but not to other proteins as judged by Western blot analysis. Using these antisera, flow cytometry analysis demonstrated that antibodies to AS(44-331) bound to a surface-exposed region of Asc10. Furthermore, antibodies specific for AS(44-331) were opsonic for E. faecalis expressing Asc10 in vitro but not for cells that did not express Asc10. New Zealand White rabbits immunized with AS(44-331) were challenged intravenously with E. faecalis cells constitutively expressing Asc10 in the rabbit model of experimental endocarditis. Highly immune animals did not show significant differences in clearance of organisms from the blood or spleen or in formation of vegetations on the aortic valve, in comparison with nonimmune animals. Although in vivo expression of Asc10 was demonstrated by immunohistochemistry, these experiments provide evidence that immunity to Asc10 does not play a role in protection from experimental infective endocarditis due to E. faecalis and may have important implications for the development of immunological approaches to combat enterococcal endocarditis.     

Spread of an Enterococcus faecalis sequence type 6 (CC2) clone in patients undergoing selective decontamination of the digestive tract

Enterococcus faecalis (E. faecalis) is a common cause of nosocomial infection in immunocompromised patients. The presence and dissemination of high‐risk clonal complexes, such as CC2, is an ongoing problem in hospitals. The aim of this work was to characterize 24 E. faecalis isolates from ICU patients undergoing selective decontamination of the digestive tract (SDD) by phenotypical (antimicrobial susceptibility) and genotypical (presence of virulence genes, RAPD‐PCR and MLST) methods. Our results showed high prevalence of the ST6 E. faecalis clone (91.6%), especially adapted to the hospital environment, with a multidrug resistance pattern and a multitude of putative virulence genes. In addition, ST179 (4.2%) and ST191 (4.2%) were detected. By RAPD–PCR analysis, the 22 isolates identified as ST6 showed six different DNA patterns, while the two remaining isolates, ST179 and ST191, showed two additional profiles. CC2 is a known clonal complex with high adaptability to hospital environment and worldwide distribution. The high prevalence of the ST6 clone in the studied population could be related to the presence of gentamicin in the SDD mixture since most strains were gentamicin resistant. Consequently, strict surveillance should be applied for rapid detection and control of this clone to prevent future spread outside the ICU.     

Recurrent bacteremia with Enterococcus faecalis,the clinical findings predicting endocarditis,and genomic characterization of the isolates: a retrospective cohort study

Multiple episodes of Enterococcus faecalis bacteremia (EfsB) may indicate a relapse and be due to an undiagnosed infective endocarditis (IE). The aims were to study the clinical presentation of patients with EfsB with focus on the risk of recurrent infection and IE, identify potential improvements of the management, and to investigate whether E. faecalis isolates from different episodes in the same patient were identical. In a retrospective study, a cohort of patients with monomicrobial (M) EfsB episodes was analyzed. Clinical data from medical records were collected. Furthermore, blood culture isolates from patients with multiple episodes were subjected to whole genome sequencing and multilocus sequence typing. In 666 episodes of MEfsB, 69 patients with IE and 43 with recurrent infections were found. Patients without IE, but with a following episode diagnosed as IE, were compared to those without a following episode. Variables significantly correlated with IE were long duration of symptoms, growth in all blood cultures, unknown origin of infection, heart murmur, and predisposition for IE. Transesophageal echocardiography, all negative, was done in 4 out of 11 episodes during the first episodes, later diagnosed with IE. In 28 of 31 patients with two or more EfsB episodes, isolates with identical sequence type were found. Episodes of EfsB in patients later diagnosed with IE showed features of IE already during the first episodes, were not adequately evaluated, are due to identical isolates, and most likely represent true relapses. Risk factor analysis should guide the use of echocardiography.

     

Nucleotide sequence of the gelatinase gene (gelE) from Enterococcus faecalis subsp. liquefaciens.

The gene coding for gelatinase (also called metalloendopeptidase II; microbial proteinase, EC 3.4.24.4) of Enterococcus faecalis subsp. liquefaciens strain OG1-10 was cloned in an Escherichia coli-Enterococcus shuttle vector, and its nucleotide sequence was determined. The DNA sequence encodes one large open reading frame (ORF) with 509 amino acid residues. The ORF contains a signal sequence in its N-terminal region, whereas the N-terminal amino acid sequence determined from the purified extracellular proteinase starts at residue 192 deduced from the ORF. This implies that the gelatinase is synthesized as a prepropolypeptide or prezymogen. The mature gelatinase contains 318 amino acid residues (molecular weight, 34,582) and has significant homology with neutral proteinases from Bacillus species and elastase from Pseudomonas aeruginosa.