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vancomycin resistance gene vanc is specific to enterococcus gallinarum.nearly all strains of enterococcus gallinarum are resistant to low levels of vancomycin. the glycopeptide resistance gene vanc from e. gallinarum bm4174 has recently been cloned and sequenced. a probe specific for vanc hybridized with a 2.7-kb ecori and a 4.5-kb hindiii fragment of total dna from the 42 strains of e. gallinarum studied. no homology was detected with dna of strains belonging to other species intrinsically resistant to vancomycin, including enterococcus casseliflavus, a species th ...19921416893
enterococcus flavescens sp. nov., a new species of enterococci of clinical origin.four yellow-pigmented group d enterococci of uncertain taxonomic position were isolated from several humans with severe infections. the results of dna composition, dna-dna hybridization, fatty acid content, and biochemical property studies demonstrated that these organisms were slightly related to other previously described yellow-pigmented enterococcal species and constitute a new species, for which we propose the name enterococcus flavescens. the type strain of e. flavescens is strain ccm 4239 ...19921503970
characterization of yellow-pigmented enterococci from severe human infections.four strains of yellow-pigmented enterococci that resembled the species enterococcus casseliflavus were isolated from patients who had undergone surgical treatment. they were substantially homologous in terms of biochemical properties, antibiotic susceptibilities, and plasmid dna profiles. yellow-pigmented enterococci could be another potentially important cause of nosocomial infection in surgical units.19911757566
use of primers selective for vancomycin resistance genes to determine van genotype in enterococci and to study gene organization in vana isolates.vancomycin resistance in enterococci is an emerging therapeutic problem. resistance is not always detected by standard microbiological methods. oligonucleotide primers for pcr were designed to target amplification of defined regions of genes of the vana cluster, as well as vanb and vanc1. these primers correctly identified 30 vancomycin-resistant isolates tested (17 vana, 7 vanb, and 6 enterococcus gallinarum). no amplification was observed with enterococcus casseliflavus or vancomycin-susceptib ...19957486917
comparison of pigment production and motility tests with pcr for reliable identification of intrinsically vancomycin-resistant enterococci.forty-eight clinical isolates identified as either enterococcus faecium or enterococcus faecalis with the microscan system (dade international, microscan inc., west sacramento, calif.) were further characterized by two supplementary biochemical tests (pigment production and motility). twenty isolates (42%), all initially identified as e. faecium, were motile. of these 20, 8 isolates (17%) produced yellow pigment and were identified as enterococcus casseliflavus and the remaining 12 (25%) were no ...19957665675
emergence of high-level resistance to glycopeptides in enterococcus gallinarum and enterococcus casseliflavus.enterococcus gallinarum bm4231 and enterococcus casseliflavus bm4232, isolated from the feces of a patient under oral therapy with vancomycin, were resistant to high levels of vancomycin (mics of > 256 micrograms/ml) and teicoplanin (mics of 128 and 64 micrograms/ml, respectively). this phenotype is new for these bacterial species that are naturally resistant to low levels of vancomycin and appears to be due to in vivo acquisition of plasmid pip218 carrying the vana gene cluster.19947979308
presumptive fecal streptococci in environmental samples characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.the use of fecal streptococci as fecal indicators requires better knowledge of the ecology of these bacteria. we isolated 371 presumptive fecal streptococci from environmental samples--domestic wastewater, forest industry wastewater, contaminated surface and seawater, well water, cow dung, bird droppings, and pristine waters--and clustered them according to their protein profiles in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. some clusters could be tentati ...19938357252
characterization of bacteriocins from enterococcus faecium with activity against listeria monocytogenes.laboratory cultures and environmental isolates of bacteria were screened for antagonism towards listeria monocytogenes using an agar spot test. seven of the 163 strains that were tested, one streptococcus bovis, one enterococcus casseliflavus, two e. avium and three e. faecium, consistently displayed antilisterial activity. cell-free, ph-neutralized supernatants prepared from the three e. faecium strains (jbl1061, jbl1083 and jbl1351) exhibited strong antilisterial activity against l. monocytoge ...19938398626
bacterial resistance to vancomycin: overproduction, purification, and characterization of vanc2 from enterococcus casseliflavus as a d-ala-d-ser ligase.the vanc phenotype for clinical resistance of enterococci to vancomycin is exhibited by enterococcus gallinarum and enterococcus casseliflavus. based on the detection of the cell precursor udp-n-acetylmuramic acid pentapeptide intermediate terminating in d-ala-d-ser instead of d-ala-d-ala, it has been predicted that the vanc ligase would be a d-ala-d-ser rather than a d-ala-d-ala ligase. overproduction of the e. casseliflavus atcc 25788 vanc2 gene in escherichia coli and its purification to homo ...19979294159
effects of different test conditions on mics of food animal growth-promoting antibacterial agents for enterococci.the influence of the addition of sheep blood to mueller-hinton ii agar and the effects of aerobic incubation with or without co2 and of anaerobic incubation were tested with bacitracin, tylosin, avoparcin, virginiamycin, avilamycin, narasin, and flavomycin on enterococci. the antibacterial activity of bambermycin (flavomycin) was strongly inhibited by the addition of blood, except with the species enterococcus faecium, enterococcus mundtii, enterococcus hirae, enterococcus casseliflavus, and ent ...19989650934
the effect of ph on the inhibition of bacterial growth by physiological concentrations of butyric acid: implications for neonates fed on suckled milk.butyric acid is released from milk by pre-intestinal lipases during suckling. it is also known to inhibit bacterial growth. to investigate whether butyric acid may be a significant factor in controlling bacterial growth in the stomach of pre-weaned animals, the ability of butyric acid to inhibit growth of selected bacteria was tested over physiological ranges of ph and butyric acid concentrations. six enteric and environmental strains of bacteria were used: two strains of escherichia coli, klebs ...19989717513
anaerobic transformation of quercetin-3-glucoside by bacteria from the human intestinal tract.from human feces two phenotypically different types of bacteria were isolated on quercetin-3-glucoside as carbon and energy source. isolates of one type were identified as strains of enterococcus casseliflavus. they utilized the sugar moiety of the glycoside, but did not degrade the aglycon further. the sugar moiety (4 mm) was fermented to 5.5 +/- 2.1 mm formate, 2.1 +/- 0.7 mm acetate, 1.6 +/- 0.3 mm l-lactate, and 1.3 +/- 0.4 mm ethanol. the second type of isolate was identified as eubacterium ...19999914304
use of tests for acidification of methyl-alpha-d-glucopyranoside and susceptibility to efrotomycin for differentiation of strains of enterococcus and some related genera.a total of 107 enterococcus strains, 10 vagococcus fluvialis strains, and 8 lactococcus garvieae strains were tested for acidification of methyl-alpha-d-glucopyranoside (mgp) and susceptibility to 100-microg efrotomycin (efro) disks. all 26 strains of enterococcus casseliflavus, including 3 nonmotile and 2 nonpigmented strains, acidified mgp and were resistant to efro. all 22 strains of enterococcus gallinarum, including 5 nonmotile strains, also acidified mgp and were resistant to efro. none of ...19989620381
acidification of methyl-alpha-d-glucopyranoside: a useful test to differentiate enterococcus casseliflavus and enterococcus gallinarum from enterococcus faecium species group and from enterococcus faecalis.enterococcus gallinarum and e. casseliflavus are difficult to differentiate from other enterococci, particularly e.faecium. the former two species were found to produce acid from methyl-alpha-d-glucopyranoside in phenol red broth, while e.faecalis strains and strains of the e.faecium species group, including e.faecium, e.durans, e. hirae, and e.mundtii, failed to produce acid from this substrate.19968880532
enterococcus casseliflavus septicaemia in a patient with acute myeloid leukaemia. 19947957279
dna hybridization and contour-clamped homogeneous electric field electrophoresis for identification of enterococci to the species level.in this study, 113 enterococcus faecium, 37 enterococcus faecalis, 24 enterococcus gallinarum, 15 enterococcus raffinosus, and 13 enterococcus casseliflavus clinical isolates and american type culture collection (atcc) strains were evaluated by contour-clamped homogeneous electric field electrophoresis. thirty-one of the e. faecium, 22 of the e. faecalis, 24 of the e. gallinarum, 15 of the e. raffinosus, and 13 of the e. casseliflavus isolates were also evaluated by dna-dna hybridization. genomi ...19957699030
investigation of the reformulated remel synergy quad plate for detection of high-level aminoglycoside and vancomycin resistance among enterococci.we investigated the accuracy of the recently released remel synergy quad plate, a commercially available agar screening method for detecting high-level aminoglycoside and vancomycin resistance among enterococci that is based on the national committee for clinical laboratory standards recommended guidelines (national committee for clinical laboratory standards, m7-a3, 1993). the synergy quad correctly determined the gentamicin and streptomycin resistance status for > or = 97% of 147 enterococcus ...19957650204
on the microbiological profile of traditional portuguese sourdough.traditional manufacture of bread from maize has been noted to play important roles from both economic and social standpoints; however, enforcement of increasingly strict hygiene standards requires thorough knowledge of the adventitious microbiota of the departing dough. to this goal, sourdough as well as maize and rye flours from several geographic locations and in two different periods within the agricultural year were assayed for their microbiota in sequential steps of quantification and ident ...199910606146
ability of commercial and reference antimicrobial susceptibility testing methods to detect vancomycin resistance in enterococci.we evaluated the abilities of 10 commercially available antimicrobial susceptibility testing methods and four reference methods (agar dilution, broth microdilution, disk diffusion, and the agar screen plate) to classify enterococci correctly as vancomycin susceptible or resistant using 50 well-characterized strains of enterococci. there was a high level of agreement of category classification data obtained with broth-based systems (sceptor, micromedia, pasco, and sensititre), agar dilution, and ...19957650179
sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci.simple pcr and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 438-bp-long dna fragment internal (soda(int)) to the soda gene encoding the manganese-dependent superoxide dismutase in 19 enterococcal type strains (enterococcus avium, enterococcus casseliflavus, enterococcus cecorum, enterococcus columbae, enterococcus dispar, enterococcus durans, enterococcus faecalis, enterococcus faecium, enterococcus flavescens, enterococcus gallinarum, enterococc ...200010618129
vancomycin susceptibility and identification of motile enterococci.thirty-seven clinical isolates of enterococcus gallinarum and enterococcus casseliflavus and three type or reference strains of the species were studied with respect to vancomycin susceptibility and key identification characteristics. with the exception of one clinical isolate of e. casseliflavus (mic, 4 micrograms/ml), mics of vancomycin were 8 to 32 micrograms/ml. the type strain of e. gallinarum, ncdo 2313, and five of the clinical isolates had similar penicillin-binding protein profiles and ...19911939593
clustered bacteremias in a hemodialysis unit: cross-contamination of blood tubing from ultrafiltrate waste.to determine the cause of clustered bacteremias occurring among chronic hemodialysis patients.19921564314
colonization and microbiology of the motile enterococci in a patient population.the motile enterococci with the vanc gene have intrinsic low-level resistance to vancomycin, but have not been implicated in a nosocomial outbreak. we determined the colonization rate of motile enterococci in hospitalized and nonhospitalized patients. perianal or stool specimens were cultured in enterococcosel broth supplemented with 6 micrograms of vancomycin per ml. rapid motility and pigment tests were performed on all enterococci isolated. a total of 82 motile and/or pigmented enterococci we ...19989764390
a new high-level gentamicin resistance gene, aph(2'')-id, in enterococcus spp.enterococcus casseliflavus uc73 is a clinical blood isolate with high-level resistance to gentamicin. dna preparations from uc73 failed to hybridize with intragenic probes for aac(6')-ie-aph(2'')-ia and aph(2'')-ic. a 4-kb fragment from uc73 was cloned and found to confer resistance to gentamicin in escherichia coli dh5alpha transformants. nucleotide sequence analysis revealed the presence of a 906-bp open reading frame whose deduced amino acid sequence had a region with homology to the aminogly ...19989593155
cloning and sequencing of two enterococcal glpk genes and regulation of the encoded glycerol kinases by phosphoenolpyruvate-dependent, phosphotransferase system-catalyzed phosphorylation of a single histidyl residue.the glpk genes of enterococcus casseliflavus and enterococcus faecalis, encoding glycerol kinase, the key enzyme of glycerol uptake and metabolism in bacteria, have been cloned and sequenced. the translated amino acid sequences exhibit strong homology to the amino acid sequences of other bacterial glycerol kinases. after expression of the enterococcal glpk genes in escherichia coli, both glycerol kinases were purified and were found to be phosphorylated by enzyme i and the histidine-containing p ...19979162046
species identification of enterococci via intergenic ribosomal pcr.accurate species identification of enterococci has become important with the wide prevalence of acquired vancomycin resistance and the presence of less epidemiologically important, inherently vancomycin-resistant enterococci. using a collection of enterococcal strains, we found that pcr amplification of the intergenic spacer (its-pcr) between the 16s and 23s rrna genes can produce amplicon profiles characteristic of the enterococcus examined. the species examined were group i enterococci (entero ...19979114380
evidence for horizontal gene transfer in evolution of elongation factor tu in enterococci.the elongation factor tu, encoded by tuf genes, is a gtp binding protein that plays a central role in protein synthesis. one to three tuf genes per genome are present, depending on the bacterial species. most low-g+c-content gram-positive bacteria carry only one tuf gene. we have designed degenerate pcr primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. the amplified dna fragments were ...200011092850
degradation of quercetin-3-glucoside in gnotobiotic rats associated with human intestinal bacteria.the two bacterial species, eubacterium ramulus and enterococcus casseliflavus, which had previously been isolated from human faeces using the flavonoid quercetin-3-glucoside as the growth substrate, were tested for their ability to utilize this compound in vivo.200011123476
unorthodox antibiotic combinations including ciprofloxacin against high-level gentamicin resistant enterococci.development of high-level gentamicin resistance among enterococci represents a serious therapeutic problem as it precludes synergy between aminoglycosides and cell-wall active agents. as part of a search for active antibiotic combinations against enterococci with high-level gentamicin resistance, we tested by the time kill curve method the efficacy of ciprofloxacin combined with ampicillin, trimethoprim-sulphamethoxazole, vancomycin or teicoplanin against ten isolates of enterococcus faecium, th ...19968722538
the vanb gene confers various levels of self-transferable resistance to vancomycin in enterococci.thirty-nine strains of enterococcus faecium and enterococcus faecalis resistant to vancomycin and susceptible to teicoplanin on disk susceptibility testing (phenotypic class b) were isolated in 15 hospitals in europe and the united states. the mics of vancomycin for these strains ranged from 4 to 1024 micrograms/ml. part of the vancomycin resistance gene vanb from e. faecalis v583 hybridized with a single but variably sized hindiii-kpni fragment of total dna from all 39 strains. this indicates t ...19938486958
in vitro activity of rp59500, an injectable streptogramin antibiotic, against vancomycin-resistant gram-positive organisms.the in vitro activity of rp59500, a streptogramin antibiotic, against 146 clinical isolates of vancomycin-resistant gram-positive bacteria was examined. five strains of the species enterococcus casseliflavus and enterococcus gallinarum, for which the mic of vancomycin was 8 micrograms/ml, were also studied. twenty-eight vancomycin-susceptible strains of enterococcus faecalis and enterococcus faecium were included for comparison. the drug was highly active against leuconostoc spp., lactobacillus ...19938460927
analysis of genes encoding d-alanine-d-alanine ligase-related enzymes in enterococcus casseliflavus and enterococcus flavescens.using degenerate oligonucleotides complementary to sequences encoding conserved amino acid motifs in d-alanine-d-alanine (ddl) ligases, we have amplified ca. 600-bp fragments from enterococcus casseliflavus atcc 25788 and enterococcus flavescens ccm439. sequence analysis of the amplification products indicated that each strain possessed two genes, ddle. cass. and vanc-2, and ddle. flav. and vanc-3, respectively, encoding ddl-related enzymes. the fragments internal to the vanc genes were 98.3% id ...19947986009
resistance of enterococcus faecium to neutrophil-mediated phagocytosis.during a previous study of the opsonic requirements for neutrophil (polymorphonuclear leukocyte [pmn])-mediated killing of enterococci, we identified two strains of enterococcus faecium (tx0015 and tx0016) that were resistant to pmn-mediated killing. to better define the mechanism of this resistance, we examined phagocytosis with a fluorescence assay and found that tx0016 was completely resistant to phagocytosis by pmns; this finding was confirmed by electron microscopy. examination of multiple ...19947960141
bile salt hydrolase activity of enterococci isolated from food: screening and quantitative determination.one hundred seventeen enterococcal strains isolated from food (47 enterococcus faecium, 48 enterococcus faecalis, 16 enterococcus durans, 2 enterococcus gallinarum, 3 enterococcus casseliflavus, and 1 enterococcus malodoratus) were screened for bile salt hydrolase (bsh) activity on de man, rogosa, and sharpe agar medium containing taurocholic acid and calcium chloride. the highest incidence of bsh-active strains was observed for e. faecalis (81%) followed by e. faecium (50%) and e. durans (44%). ...200111348009
a pseudoepidemic due to laboratory contamination deciphered by molecular analysis.a clinical, microbiological, and molecular analysis to identify the source of a cluster of pseudoinfections.19957759823
streptococcus faecium var. casselifavus, nov. var.streptococcus faecium var. casseliflavus is a gram-positive, spherical cell. the cells occur chiefly as pairs within chains and elongate to ogive-shaped cells during growth. growth is good on 5% bile salts-agar and in broth at 10 c, and in broth adjusted to ph 9.6 or containing 6.5% nacl, but many strains fail to grow at 45 c. litmus is reduced rapidly prior to formation of an acid curd. few strains release ammonia from arginine or serine. the organism is not proteolytic and does not produce h(2 ...19685669887
practical approach to the identification of clinically relevant enterococcus species.enterococci have become important nosocomial pathogens, with enterococcus faecalis and then enterococcus faecium predominating. because of the emergence of glycopeptide (vancomycin and teicoplanin) resistance in enterococci, laboratories have been required to screen for resistant strains and to identify them to the species level. this has resulted in the need for accurate identification of species less commonly associated with clinical infections, such as enterococcus casseliflavus and enterococ ...199910403095
augmented production of extracellular superoxide by blood isolates of enterococcus faecalis.to assess the frequency of extracellular superoxide (o-2) production by enterococci, multiple species were surveyed in a whole organism assay for their ability to reduce ferricytochrome c in a superoxide dismutase-inhibitable fashion. for stool and clinical enterococcal isolates and 12 type strains, only enterococcus faecalis (87/91 isolates and atcc 19433), enterococcus faecium (5/13 isolates), enterococcus casseliflavus (atcc 25778), and enterococcus gallinarum (atcc 35038) produced extracellu ...19968627044
enterococci in insects.enterococci were obtained from 213 of 403 insects cultured during a 14-month period, in numbers from 10(3) to 3 x 10(7)/g of insect. insects were taken only from nonurban, wild, and cultivated fields and woods. in species of insects carrying them, enterococci were not always present in every individual cultured, and often more than one species of enterococcus occurred within a species. enterococci were obtained from certain insects taken in the field during the dormant season, suggesting their r ...19724628796
evaluation of arbitrarily primed pcr analysis and pulsed-field gel electrophoresis of large genomic dna fragments for identification of enterococci important in human medicine.the increasing problems encountered with enterococcal nosocomial infections and the intrinsic and acquired resistance of the enterococci to different antimicrobial compounds highlight the need for a rapid identification technique. enterococcus faecalis is readily identified by biochemical tests, but species differentiation within the enterococcus faecium and enterococcus gallinarum species groups is less well established. in the present study, 66 strains representing the most prevalent human ent ...19979103648
comparison of agar-based media for primary isolation of glycopeptide-resistant enterococci.objective: to compare four vancomycin-containing agar media for the isolation of glycopeptide-resistant enterococci (gre) from clinical fecal specimens: kanamycin---aesculin---azide (kaa) agar; bile---aesculin---polymixin (bap) agar; aztreonam---amphotericin blood (cbaa) agar; and neomycin blood (cbn) agar. methods: fecal specimens from 125 patients were inoculated onto each medium. media were examined for enterococci after incubation for up to 48 h. enterococci were identified to species level, ...199711864182
characterization of yellow-pigmented and motile enterococci isolated from intestines of the garden snail helix aspersa.enterococci associated with garden snails (helix aspersa) were studied in order to obtain reliable species identification and characterization.200211972701
vancomycin-resistant enterococci (vre) colonization of high-risk patients in tertiary care canadian hospitals. canadian vre surveillance group.we isolated 1487 enterococcus species from 1200 stool specimens collected from high-risk patients in 12 canadian tertiary care hospitals between october 1995 and november 1996. the composition of the 1487 isolates was 601 vancomycin-sensitive enterococcus faecalis (40.4%), 667 vancomycin-sensitive enterococcus faecium (44.9%), 18 vancomycin-resistant (nine isolates mic 8-16 micrograms/ml; nine isolates mic > or = 32 micrograms/ml) e. faecium (vref) (1.2%), 95 vancomycin-sensitive enterococcus ga ...199910529874
heterogeneity of resistance elements in clinical isolates of enterococci with high-level gentamicin resistance.high-level resistance (minimum inhibitory concentration, mic > 1,000 micrograms/ml) to gentamicin (hlgr) in enterococci is common in taiwan. in this study, we investigated the distribution of gentamicin resistance elements in enterococci isolated at national taiwan university hospital in a 1-year period, and also examined the transfer and the genetic variability of the resistance elements of different isolates. among 109 isolates tested, 43 (39%) hlgr isolates were identified. hlgr was most comm ...19989884489
effect of vancomycin on intestinal flora of patients who previously received antimicrobial therapy.to evaluate the ecological disturbances of peroral vancomycin administration following cephalosporin administration, 20 healthy volunteers received cefuroxime axetil tablets (250 mg) perorally twice a day for 1 week, and 10 of these volunteers subsequently received vancomycin capsules (125 mg) perorally four times daily for 7 days. the concentration of vancomycin in feces after 1 week of vancomycin administration was high (mean +/- sd, 520 +/- 197 mg/kg), which correlated with the ecological dis ...19979314469
species identities of enterococci isolated from clinical specimens.conventional tests and commercially available systems were used to determine the species identities of clinical isolates of enterococci. strict adherence to the conventional test scheme of facklam and collins (r. r. facklam and m. d. collins, j. clin. microbiol. 27:731-734, 1989) resulted in the misidentification of lactose-negative enterococcus faecalis isolates as enterococcus solitarius, but this problem was overcome by the application of additional tests. the commercially available systems t ...19902108992
in vitro activity of a new cephalosporin, rwj-54428, against streptococci, enterococci and staphylococci, including glycopeptide-intermediate staphylococcus aureus.the need for new antimicrobial agents with activity against gram-positive organisms has become increasingly important because of emerging resistance. we compared the activity of a new b-lactam antimicrobial agent, rwj-54428 (mc-02 479), with representatives of other classes of antimicrobial agents against 76 staphylococcus aureus (including four glycopeptide- intermediate strains), 50 coagulase-negative staphylococci, 20 enterococcus faecalis, 20 enterococcus faecium, 10 enterococcus gallinarum/ ...200212003982
source of enterococci in a farmhouse raw-milk cheese.enterococci are widely distributed in raw-milk cheeses and are generally thought to positively affect flavor development. their natural habitats are the human and animal intestinal tracts, but they are also found in soil, on plants, and in the intestines of insects and birds. the source of enterococci in raw-milk cheese is unknown. in the present study, an epidemiological approach with pulsed-field gel electrophoresis (pfge) was used to type 646 enterococcus strains which were isolated from a ch ...200212089042
enterococcal-type glycopeptide resistance genes in non-enterococcal organisms.although the emergence of vancomycin-resistant enterococci can be attributed, in part, to the increasing use of vancomycin in clinical practice, and glycopeptide use in animal husbandry, the origins of the enterococcal vancomycin resistance genes are not clear. the vancomycin resistance-associated genes in enterococcus gallinarum, enterococcus casseliflavus/flavescens, lactobacillus spp., leuconostoc spp., pediococcus spp., and erysipelothrix rhusiopathiae, are not the source of the high-level v ...200010731599
random amplification of polymorphic dna versus pulsed field gel electrophoresis of smai dna macrorestriction fragments for typing strains of vancomycin-resistant enterococci.genetic typing of vancomycin-resistant enterococci (vre) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. we here compare random amplification of polymorphic dna (rapd) analysis with pulsed field gel electrophoresis (pfge) of dna macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. for the latter method, empirical guidelines for the interpretation of the dna finger ...200011040427
identification of enterococcus species and phenotypically similar lactococcus and vagococcus species by reverse checkerboard hybridization to chaperonin 60 gene sequences.data from four recent studies (s. h. goh et al., j. clin. microbiol. 36:2164-2166, 1998; s. h. goh et al., j. clin. microbiol. 34:818-823, 1996; s. h. goh et al., j. clin. microbiol. 35:3116-3121, 1997; a. y. c. kwok et al., int. j. syst. bacteriol. 49:1181-1192, 1999) suggest that an approximately 600-bp region of the chaperonin 60 (cpn60) gene, amplified by pcr with a single pair of degenerate primers, has utility as a potentially universal target for bacterial identification (id). this cpn60 ...200011060051
diversity of domain v of 23s rrna gene sequence in different enterococcus species.the highly conserved central loop of domain v of 23s rna (nucleotides 2042 to 2628; escherichia coli numbering) is implicated in peptidyltransferase activity and represents one of the target sites for macrolide, lincosamide, and streptogramin b antibiotics. dna encoding domain v (590 bp) of several species of enterococcus was amplified by pcr. twenty enterococcal isolates were tested, including enterococcus faecium (six isolates), enterococcus faecalis, enterococcus avium, enterococcus durans, e ...200011060057
effect of raw-milk cheese consumption on the enterococcal flora of human feces.enterococci are one of the major facultative anaerobic bacterial groups that reside in the human gastrointestinal tract. in the present study, the composition of the enterococcal fecal flora in three healthy humans was analyzed before, during, and after the daily consumption of approximately 125 g of a raw-milk cheddar-type cheese containing 3.2 x 10(4) enterococci/g of cheese. enterococcal counts ranged between 1.4 x 10(2) and 2.5 x 10(8) cfu/g of feces and differed from subject to subject and ...200312514010
prevalence of vancomycin-resistant enterococci in europe.the aim of the present study was to determine the prevalence of vancomycin-resistant enterococci (vre) in europe. overall, 49 laboratories in 27 countries collected 4,208 clinical isolates of enterococci. species identification, susceptibility testing, and van gene determination by polymerase chain reaction were performed in a central laboratory. overall, 18 vana and 5 vanb isolates of vre were found. the prevalence of vana vre was highest in the uk (2.7%), while the prevalence of vanb vre was h ...200011152305
species prevalence and antibacterial resistance of enterococci isolated in kuwait hospitals.this study investigated the species prevalence and antibacterial resistance among enterococci isolated in kuwait hospitals. they consisted of 415 isolates of enterococcus faecalis (85.3 %), enterococcus faecium (7.7 %), enterococcus casseliflavus (4.0 %), enterococcus avium (1.2 %), enterococcus durans (1.0 %), enterococcus gallinarium (0.5 %) and enterococcus bovis (0.2 %) isolated from urine (36.6 %), blood (10.4 %), wound swabs (11.0 %), stool samples (12.0 %), high vaginal swabs (9.0 %), end ...200312543923
in-vitro activity of fosfomycin against vancomycin-resistant enterococci.the effect of fosfomycin against 69 vancomycin-resistant isolates of enterococcus faecium (vana), five of e. faecium (vanb), 11 of enterococcus faecalis (vana), three of e. faecalis (vanb), 10 of enterococcus gallinarum (vanc1) and two of enterococcus casseliflavus (vanc2) and glycopeptide-sensitive e. faecium (n = 8) and e. faecalis (n = 10) was tested in vitro. fosfomycin inhibited 97%, 94% and 96% of the vancomycin-resistant strains, according to results of agar dilution, broth microdilution, ...199911252326
glycerol transport and phosphoenolpyruvate-dependent enzyme i- and hpr-catalysed phosphorylation of glycerol kinase in thermus flavus.the genes glpk and glpf, encoding glycerol kinase and the glycerol facilitator of thermus flavus, a member of the thermus/deinococcus group, have recently been identified. the protein encoded by glpk exhibited an unusually high degree of sequence identity (80-6%) when compared to the sequence of glycerol kinase from bacillus subtilis and a similar high degree of sequence identity (64.8%) was observed when the sequences of the glycerol facilitators of the two organisms were compared. the work pre ...199910589729
emergence of vancomycin-resistant enterococci at a university hospital in taiwan: persistence of multiple species and multiple clones.to describe the epidemiology of vancomycin-resistant enterococci (vre) in a university hospital in taipei, taiwan.199910614607
[epidemiological study on vancomycin-resistant enterococci from fecal samples in the east area of japan].recently, vancomycin-resistant enterococci (vre) have become important nosocomial pathogens in the world. in japan, the vre-infection was first reported in 1996. however, an epidemiological study on vre has not been aggressively done in japan. we conducted a survey study to explore the incidence and antimicrobial susceptibility of vancomycin-resistant enterococci isolated from fecal samples at 45 hospitals in the east area of japan (kanto, koshinetsu, tohoku, and hokkaido) during june 1998 to ma ...200111521276
mechanism of metronidazole-resistance by isolates of nitroreductase-producing enterococcus gallinarum and enterococcus casseliflavus from the human intestinal tract.enterococcus casseliflavus and enterococcus gallinarum strains resistant to metronidazole, nitrofurantoin and nitrofurazone were isolated from fecal samples of a patient with recurrent ulcerative colitis treated with metronidazole. unlike other metronidazole-resistant bacteria, these strains produced nitroreductase but metabolized metronidazole to compounds that could not be detected by liquid chromatography with uv or mass spectral analysis. metronidazole-susceptible clostridium perfringens gre ...200312951241
active-site mutants of the vanc2 d-alanyl-d-serine ligase, characteristic of one vancomycin-resistant bacterial phenotype, revert towards wild-type d-alanyl-d-alanine ligases.the rising number of vancomycin-resistant enterococci (vres) is a major concern to modern medicine because vancomycin is currently the 'last resort' drug for life-threatening infections. the d-alanyl-d-x ligases (where x is an hydroxy or amino acid) of bacteria catalyze a critical step in bacterial cell-wall peptidoglycan assembly. in bacteria that produce glycopeptide antibiotics and in opportunistic pathogens, including vres, d-, d-ligases serve as switches that confer antibiotic resistance on ...19989545431
antimicrobial susceptibility and species identification for clinical isolates of enterococci.in order to describe the susceptibility patterns and determine the identities of enterococcus spp. isolated at a regional teaching hospital in taichung during the period from june through november 1998, 96 clinical isolates of enterococci were collected for further analysis. major sources of these isolates included urine, wound, and pus. species identification was performed using the api 20 strep system and supplemental tests. the minimum inhibitory concentrations (mics) of six antimicrobial age ...200011269371
clinical and epidemiological features of enterococcus casseliflavus/flavescens and enterococcus gallinarum bacteremia: a report of 20 cases.the clinical significance of intrinsically vancomycin-resistant enterococci is not yet fully established, as these organisms are infrequently recovered from clinical specimens. we report our experience with 20 cases of enterococcus gallinarum and enterococcus casseliflavus/flavescens bacteremia in humans from 1992 through 1998. sixteen cases of bacteremia were caused by e. gallinarum. underlying conditions were present in 19 (95%) of the patients and included malignancy, receipt of transplant, a ...200111340524
enterococcus casseliflavus bacteremia. case report and literature review.motile enterococci are rare causes of enterococcal bacteremia, usually in immunocompromised patients. we describe a case of polymicrobial bacteremia, including enterococcus casseliflavus, escherichia coli, and morganella morganii, in an 80-year old woman, shortly after a liver biopsy performed to exclude cholangiocarcinoma. we further discuss the unique genetic characteristics and the clinical significance of such isolates, such as their intrinsic resistance to vancomycin, and the low virulence ...200414720498
identification of enterococcal isolates by temperature gradient gel electrophoresis and partial sequence analysis of pcr-amplified 16s rdna variable v6 regions.based on partial sequence analysis of pcr-amplified 16s rdna variable v6 regions of 14 enterococcal type strains, enterococcus faecalis, enterococcus mundtii, enterococcus gallinarum, enterococcus avium, enterococcus raffinosus and enterococcus saccharolyticus showed characteristic sequence motifs which made it possible to separate them into six individual species lines. furthermore, two species cluster groups could be identified, including (i) enterococcus faecium, enterococcus durans, enteroco ...200111430498
the in vitro activity of daptomycin against 514 gram-positive aerobic clinical isolates.the in vitro activity of daptomycin was assessed in comparison with that of vancomycin and penicillin against a wide range of gram-positive aerobic clinical isolates. mics were determined by an agar dilution method on mueller-hinton agar (nccls/eucast) and on isotonic agar adjusted to contain 50 mg/l free calcium (bsac). both media were enriched with 5% horse blood for fastidious organisms. daptomycin mics for all 172 staphylococci, including methicillin-susceptible and methicillin-resistant sta ...200111481291
study of the ecology of fresh sausages and characterization of populations of lactic acid bacteria by molecular methods.in this study, a polyphasic approach was used to study the ecology of fresh sausages and to characterize populations of lactic acid bacteria (lab). the microbial profile of fresh sausages was monitored from the production day to the 10th day of storage at 4 degrees c. samples were collected on days 0, 3, 6, and 10, and culture-dependent and -independent methods of detection and identification were applied. traditional plating and isolation of lab strains, which were subsequently identified by mo ...200415066777
comparison of simple and rapid methods for identifying enterococci intrinsically resistant to vancomycin.three different methodologies, reduction of litmus milk (lm) and acidification of arabinose (ara), acidification of methyl-alpha-d-glucopyranoside (mgp), and rapid motility (rm), for differentiating isolates of enterococcus casseliflavus and enterococcus gallinarum (intrinsically vancomycin-resistant enterococci [ivre]) from enterococcus faecalis and enterococcus faecium were evaluated. all 33 isolates of e. faecalis tested reduced lm within 4 h and were negative in all other tests, while the 53 ...19999986863
the soluble alpha-glycerophosphate oxidase from enterococcus casseliflavus. sequence homology with the membrane-associated dehydrogenase and kinetic analysis of the recombinant enzyme.the soluble flavoprotein alpha-glycerophosphate oxidase from enterococcus casseliflavus catalyzes the oxidation of a "non-activated" secondary alcohol, in contrast to the flavin-dependent alpha-hydroxy- and alpha-amino acid oxidases. surprisingly, the alpha-glycerophosphate oxidase sequence is 43% identical to that of the membrane-associated alpha-glycerophosphate dehydrogenase from bacillus subtilis; only low levels of identity (17-22%) result from comparisons with other fad-dependent oxidases. ...19989726992
mannan-degrading enzymes purified from the crop of the brown garden snail helix aspersa müller (gastropoda pulmonata).two mannan-degrading enzymes were purified from the crop of the terrestrial snail helix aspersa müller. the crude extracts were taken from dormant (for 4 months) snails. the enzymes were a betad-mannanase of 37.4 +/- 0.3 kda (ec 3.2.1.78) and a betad-mannosidase of 77.8 +/- 1.9 kda (ec 3.2.1.25). both enzymes degraded insoluble mannan, releasing either mannose only (beta-mannosidase) or oligosaccharides, possibly mannotriose and mannopentaose (beta-mannanase). the beta-mannanase had a typical en ...200111471142
characterization of high-level aminoglycoside-resistant enterococci in kuwait hospitals.this study investigated the distribution of genes for aminoglycoside-modifying enzymes (ame) and the genetic relatedness of high-level aminoglycoside-resistant enterococci isolated in kuwait hospitals. a total of 117 enterococci, consisting of 109 enterococcus faecalis, seven enterococcus faecium, and one enterococcus casseliflavus were studied. the mics of gentamicin, kanamycin, amikacin, tobramycin, and streptomycin were determined by agar dilution and the genes encoding the aac(6')- aph(2"), ...200415256029
colonization by vancomycin-resistant enterococci of the intestinal tract of patients in intensive care units from french general hospitals.objective: to evaluate the prevalence of fecal carriage of vancomycin-resistant enterococci (vre) by patients hospitalized in intensive care units from 24 french general hospitals. methods: rectal swabs were obtained from 647 patients hospitalized in intensive care units during the month of june 1994 and plated on agar medium selective for vancomycin-resistant enterococci. the glycopeptide resistance phenotypes and genotypes of the enterococci detected were characterized. results: thirty-two of ...199711864101
susceptibility to quinupristin/dalfopristin and other antibiotics of vancomycin-resistant enterococci from the uk, 1997 to mid-1999.susceptibility to quinupristin/dalfopristin and other antibiotics was studied for clinical isolates of vancomycin-resistant enterococci (vre) referred by uk hospitals between january 1997 and june 1999. single isolates of vre from 858 patients in 136 hospitals were received, of which 76% were enterococcus faecium and 21% were enterococcus faecalis, the remainder comprising minor species. most isolates were multi-resistant. after allowing for the effect of blood, which raised the mics of quinupri ...200010882701
cost-effective algorithm for detection and identification of vancomycin-resistant enterococci in surveillance cultures.a study was undertaken to develop an easy-to-use and cost-effective algorithm for the detection and identification of vancomycin-resistant enterococci (vre) in surveillance cultures, because the incidence of vre outbreaks in institutions across canada has made continuous surveillance a necessity. enterococcus faecium and enterococcus faecalis carry transferable resistance genes and hence are a problem for infection control. in laboratory surveillance, however, enterococcus gallinarum and enteroc ...200010898139
nucleotide substitutions in vanc-2 gene of enterococcus casseliflavus isolates obtained from chickens.dna sequencing of the vanc-2 gene was partially carried out on 10 isolates of enterococcus casseliflavus obtained from 8 samples of imported chickens in japan between july 1999 and june 2001 to evaluate the variation in the gene. forty nucleotide substitutions in 36 codons were identified within 345 base pairs when compared with the vanc-2 sequence of the reference strain e. casseliflavus atcc25788. identical nucleotide substitutions were commonly found in the isolates recovered from chickens im ...200212403118
characterization of tn1546 in vancomycin-resistant enterococcus faecium isolated from canine urinary tract infections: evidence of gene exchange between human and animal enterococci.thirty-five enterococcal isolates were recovered from dogs diagnosed with urinary tract infections at the michigan state university veterinary teaching hospital over a 2-year period (1996 to 1998). isolated species included enterococcus faecium (n = 13), enterococcus faecalis (n = 7), enterococcus gallinarum (n = 11), and enterococcus casseliflavus (n = 4). antimicrobial susceptibility testing revealed several different resistance phenotypes, with the majority of the enterococcal isolates exhibi ...200212454168
enumeration and speciation of enterococci found in marine and intertidal sediments and coastal water in southern california.to determine the levels and species distribution of enterococci in intertidal and marine sediments and coastal waters at two beaches frequently in violation of bacterial water standards.200516108802
enterocin 416k1, an antilisterial bacteriocin produced by enterococcus casseliflavus im 416k1 isolated from italian sausages.enterococci (118) from italian sausages were tested for the production of antimicrobial substances. of these, 7.6% showed antibacterial activity against one or several closely related microorganisms used as indicators. enterococcus casseliflavus im 416k1 in particular produced a bacteriocin (enterocin 416k1) with strong anti-listerial antagonistic activity. the bacteriocin withstood heating at 90 degrees c for 120 min and storage at 4 degrees c for 6 months. the mode of action was identified as ...200211999113
microbiological study of lactic acid fermentation of caper berries by molecular and culture-dependent methods.fermentation of capers (the fruits of capparis sp.) was studied by molecular and culture-independent methods. a lactic acid fermentation occurred following immersion of caper berries in water, resulting in fast acidification and development of the organoleptic properties typical of this fermented food. a collection of 133 isolates obtained at different times of fermentation was reduced to 75 after randomly amplified polymorphic dna (rapd)-pcr analysis. isolates were identified by pcr or 16s rrna ...200516332762
in vitro activity of daptomycin against vancomycin-resistant enterococci of various van types and comparison of susceptibility testing methods.the increasing prevalence of vancomycin-resistant enterococcal (vre) infections and the limited number of antimicrobial agents for their treatment emphasize a need for new, more effective agents. in this study, the in vitro activity of daptomycin was determined against a collection of 156 vre from seven different institutions. van types were characterized by pcr, and pulsed-field gel electrophoresis was performed to exclude isolates with >85% relatedness by dendrogram. included were 126 enteroco ...200314638478
partial characterization of enterocin mr99 from a corn silage isolate of enterococcus faecalis.to assess the inhibitory activity on gram-positive and gram-negative bacteria of several species of enterococci recovered from a natural corn silage.200616405692
phenotypes and genotypes of vancomycin-resistant enterococci isolated during long-term follow-up in a patient with recurrent bacteremia and colonization.twenty-seven isolates of vancomycin-resistant enterococci were obtained at monthly intervals from a bed-ridden man with hypoxic encephalopathy. during the 28-month period of the patient's hospitalization, 3 episodes of bacteremia and one episode of catheter-related infection caused by vancomycin-resistant enterococci occurred. rectal swabs showed colonization of vancomycin-resistant enterococci for more than 2 years. three months after termination of antimicrobial therapy, the rectal colonizatio ...200212542250
structures of enterococcal glycerol kinase in the absence and presence of glycerol: correlation of conformation to substrate binding and a mechanism of activation by phosphorylation.the first structure of a glycerol kinase from a gram-positive organism, enterococcus casseliflavus, has been determined to 2.8 a resolution in the presence of glycerol and to 2.5 a resolution in the absence of substrate. the substrate-induced closure of 7 degrees is significantly smaller than that reported for hexokinase, a model for substrate-mediated domain closure that has been proposed for glycerol kinase. despite the 78% level of sequence identity and conformational similarity in the cataly ...200414717590
enterococcus casseliflavus septicaemia in a preterm neonate.enterococcus casseliflavus is a yellow-pigmented, motile, gram-positive coccus that is only rarely isolated from clinical specimens. we report the first case of neonatal sepsis due to e. casseliflavus in a premature neonate. precise identification of enterococcus species is difficult but is of significant clinical interest because of the intrinsic low-level resistance to vancomycin of species such as e. casseliflavus and e. gallinarum.200212160178
[a novel plasmid-mediated aminoglycosides-modifying enzyme gene, aph (2'')-ie, in a strain of high-level gentamicin resistant enterococcus casseliflavus].to identify the aminoglycoside resistance gene in the high-level gentamicin resistant (hlgr) enterococcus and its transmission mechanism.200616681903
comparison of two commercially available selective media to screen for vancomycin-resistant enterococci.campylobacter medium (campy, becton dickinson [bd] microbiology systems, cockeysville, md) and vancomycin screen agar (vsa, bd) were compared for their ability to screen for vancomycin-resistant enterococcus (vre) from primary plates. a microdilution minimal inhibitory concentration (mic) assay (pasco, bd) served as the reference vancomycin mic. in the random sample of 200 enterococcal clinical isolates tested, the distribution of isolates was as follows: enterococcus faecium, 113 (97 vre); ente ...200211789720
study of two bacteriocins produced by enterococcus casseliflavus and ent. faecalis.the antimicrobial activity of two plasmid-borne bacteriocins produced by enterococcus casseliflavus im 416k1 and ent. faecalis im 388c and their mating transferability were studied.200414746539
vancomycin-resistant enterococci isolates from the rumen content of deer.the total enumeration of enterococci from the rumen content of deer, their species determination, resistance to antibiotics with the predominant importance of vancomycin resistance, and their metabolic properties, were examined. enterococci (2.0 x 10(2) cfu per ml) encountered from the rumen of deer were phenotypically allotted to the species enterococcus casseliflavus (26.6%), e. gallinarum (26.6%) and e. faecium (6.6%). the other isolates were not phenotypically specified. although the resista ...199910453484
[characteristics of enterococcus strains isolated from neutropenic patients at the national bone-marrow transplantation center of tunis].the frequency of digestive colonization of neutropenic patients by enterococci during the phase of pre-transplant and post-transplant of bone marrow is important indeed as 441 enterococcus spp strains have been isolated from stool-cultures and other specimens whithin a period of 35 months in 80 patients. a quantitative stool culture was done on appropriate media. simple bile-esculine agar (be) and bile esculine-agar additionned with 6 mg/l of vancomycine (bev) were used for detecting enterococci ...200415255347
molecular study of vancomycin-resistant enterococci isolated from humans and from food in a cattle-rearing area of france.study possible links between vancomycin-resistant enterococci strains isolated from human stool samples and from pork or poultry food products.200415175270
lack of correlation between phenotypic techniques and pcr-based genotypic methods for identification of enterococcus spp.a total of 123 genetically-unrelated strains of enterococcus spp. strains (51 enterococcus faecalis, 57 enterococcus faecium, 10 enterococcus gallinarum, and 5 enterococcus casseliflavus) were phenotypically identified by biochemical profiles and by using an automated method. the strains were also analyzed by a pcr assay to assess the accuracy of the phenotypically-based methods for identification of enterococcus spp. with this aim, a pcr assay using different cell targets, which allows simultan ...200415246503
species distribution and antibiotic resistance patterns of enterococci isolated from food of animal origin in germany.presently, enterococci take the third place of bacterial pathogens associated with nosocomial infections, after staphylococci and escherichia coli. especially, the resistances of enterococci to several available antibiotics are threatening. we attempted to determine which species of enterococci could be found in food of animal origin and their significance according to their antibiotic resistances for human beings. from november 2000 to may 2002 we investigated 155 samples of food of animal orig ...200314597003
dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in ghana.the ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. a culture-dependent (plating and incubation, followed by repetitive-sequence-based pcr analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [dgge] of 16s rrna gene amplicons, pcr-dgge) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (lab) an ...200717277227
molecular identification and diversity of enterococci isolated from slovak bryndza cheese.three hundred and eight presumed enterococcal isolates were recovered from bryndza, a soft sheep milk cheese. the cheese samples were obtained from five different commercial distributors in slovakia and were taken at three different seasonal intervals. all isolates were identified to the species level using genotypic tools. species-specific pcr using ddl genes highlighted the predominance of enterococcus faecium (176 isolates) and assigned 50 isolates to the species enterococcus faecalis. the re ...200617325446
occurrence of vancomycin-resistant enterococci in humans and animals in the czech republic between 2002 and 2004.in the period between september 2002 and may 2004, a total of 6023 rectal swabs from humans in the czech republic were evaluated and 821 enterococcus spp. strains were isolated. nine strains (1.1 %) were identified as vancomycin-resistant enterococci (vre). two strains were vana enterococcus faecium, one strain was vanb enterococcus faecalis and six strains were vanc enterococcus casseliflavus. in total, 527 enterococcus spp. strains were isolated from poultry breeds of which 11 (2.1 %) were vre ...200516157551
vancomycin-resistant enterococci, colonizing the intestinal tract of patients in a university hospital in greece.determine the prevalence of vancomycin-resistant enterococci (vre) colonizing the intestinal tract of hospitalized patients and define risk factors.200617568849
reclassification of enterococcus flavescens pompei et al. 1992 as a later synonym of enterococcus casseliflavus (ex vaughan et al. 1979) collins et al. 1984 and enterococcus saccharominimus vancanneyt et al. 2004 as a later synonym of enterococcus italicus fortina et al. 2004.the taxonomic relatedness between the species enterococcus casseliflavus and enterococcus flavescens and between enterococcus italicus and enterococcus saccharominimus was investigated. literature data had already indicated the synonymy between e. casseliflavus and e. flavescens, but this observation had not been formally published. additional evidence that the two taxa represent a single species was provided by comparison of the partial sequences for three housekeeping genes, phenylalanyl-trna ...200616449449
clinical features and outcomes of bacteremia caused by enterococcus casseliflavus and enterococcus gallinarum: analysis of 56 cases.the clinical significance and virulence potential of enterococcus casseliflavus/flavescens and enterococcus gallinarum are still uncertain. we retrospectively analyzed 56 cases of significant bacteremia caused by e. casseliflavus or e. gallinarum. of these cases, 25 (44.6%) were associated with polymicrobial bacteremia, and 43 (76.8%) were associated with entry via the biliary tract. resistance to vancomycin was observed in 17 (30.4%) of these 56 patients, and this resistance was significantly a ...200414679448
antibody binding to streptococcus mitis and streptococcus oralis cell fractions.to determine which cell fraction(s) of streptococcus mitis biovar 1 serve as the best source of antigens recognized by salivary siga antibodies in infants.200817904095
[endocarditis due to enterococcus casseliflavus]. 200312732121
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